Antitumor vaccination using allogeneic tumor cells expressing alpha (1,3)-galactosyltransferase

ABSTRACT

The invention relates to methods and compositions for causing the selective targeting and killing of tumor cells. Through ex vivo gene therapy protocols tumor cells are engineered to express an α(1,3)galactosyl epitope. The cells are then irradiated or otherwise killed and administered to a patient. The α-galactosyl epitope causes opsonization of the tumor cell enhancing uptake of the opsonized tumor cell by antigen presenting cells which results in enhanced tumor specific antigen presentation. The animal&#39;s immune system thus is stimulated to produce tumor specific cytotoxic cells and antibodies which will attack and kill tumor cells present in the animal.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation in part of co-pending commonly ownedU.S. application Ser. No. 10/682,178 filed Oct. 9, 2003 which is acontinuation of U.S. provisional application 60/417,343 filed Oct. 9,2002. The disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatingcancer by stimulating humoral and cellular immune responses againsttumor cells. In particular, this invention is directed to methods forstimulating complement-mediated destruction of tumors cells andconcomitant stimulation of tumor specific antibody production and tumorspecific cytotoxic cells.

BACKGROUND OF THE INVENTION

A primary barrier to xenotransplantation has been the essentiallyimmediate recognition of carbohydrate epitopes present in the foreigntissue causing hyperacute xenograft rejection (HAR). The reaction beginsimmediately upon reperfusion, and once initiated destroys the foreigntissue within minutes to a few hours. The presence of HAR in somedonor/recipient combinations while not others is postulated to berelated to two primary factors, a) the binding of xenoreactive naturalantibodies of the recipient to antigens or endothelial cells in thegraft and b) the incompatibility of complement regulatory proteins inthe transplant with the complement system of the recipient, allowinguncontrolled activation of complement. Greater than 1% of thecomplement-fixing natural antibodies in human serum recognize a singlestructure-Galα(1-3)Galβ(1,4)G1cNAc-R. The synthesis ofGalα(1-3)Galβ(1,4)G1cNAc-R is catalyzed by the enzyme α(1,3)galactosyltransferase (αGT).

This enzyme catalyzes the synthesis of α-galactosyl (αGal) epitopes inthe Golgi apparatus of cells from various non-primate mammals by thefollowing reaction:

Galβ(1,4)GlcNAc-R+UDP-Gal→Galα(1-3)Ga1β(1,4)GlcNAc-R

This enzyme was found to be active in new world monkeys but not in oldworld monkeys and humans. The αGT cDNA has been cloned from bovine andmurine cDNA libraries. Larson, R. D. et al. (1989) “Isolation of a cDNAEncoding Murine UDP galactose; β-D-galactosyl-(1,4) NAcetyl-D-Glucosamine α-(1,3) Galactosyl Transferase Expression Cloningby Gene Transfer”, PNAS, USA 86:8227; and Joziasse, D. H. et al., (1989)“Bovine α-(1,3) Galactosyl Transferase: Isolation and Characterizationof a cDNA Clone, Identification of Homologous Sequences in Human GenomicDNA”, J. Biol Chem 264:14290.

The gene is present in the human genome, although no transcription hasbeen detected. Instead, two frame shift mutations were found (deletionsgenerating premature stop codons) in the human exons encoding theenzyme. See generally, Galili, Uri “Evolution in Pathophysiology of theHuman Natural anti-α-Galactosyl IgG (anti-αGal) Antibody”, SpringerSemin. Immunopathol. (1993) 15:155-171.

anti-αGal, a naturally occurring antibody present in all humans,specifically interacts with the carbohydrate epitopeGalα(1-3)Galβ(1,4)GlcNAc-R (αGal epitope). This antibody does notinteract with any other known carbohydrate epitope produced by mammaliancells (Galili, 1993, Springer Seminar Immunopathology 15:153). anti-αGalconstitutes approximately 1% of circulating IgG (Galili et al., 1984, J.Exp. Med. 160:1519) and is also found in the form of IgA and IgM (Davineet al., 1987, Kidney Int. 31:1132; Sandrin et al., 1993, Proc. Natl.Acad. Sci. USA 90:11391). It is produced by 1% of circulating Blymphocytes (Galili et al., 1993, Blood 82:2485). Production of thisnatural anti-αGal Ab in humans is constantly stimulated by the presenceof αGal carbohydrate residues present in intestinal and pulmonarybacterial flora. In humans anti-αGal reacts to the presence of thisepitope in hyperacute xenograft rejection and complement is swift andcertain resulting in destruction of foreign tissues in minutes to hours.

It is an object of this invention to develop a therapeutic cancervaccine by introducing the gene encoding for αGT into tumor cells, todrive the addition of αGal epitopes to such vaccine tumor cells in orderto allow for enhanced opsonization of the vaccine cells by naturalanti-αGal antibodies and stimulate tumor antigen presentation to inducea humoral and cellular immune response to the tumor specific antigens.

It is a further object of this invention to provide a therapeuticpharmaceutical composition comprising recombinant tumor cells whichexpress and process αGT to engineer αGal epitopes on cells.

It is a further object of the invention to provide compositions andmethods for treatment of tumors, viruses, neoplastic cells or othercells, which grow and evade the cellular and humoral immune response.

Other objects of the invention will become apparent from the descriptionof the invention which follows.

SUMMARY OF THE INVENTION

This invention relates to methods and compositions for causing an immuneresponse to selectively targeting and killing of tumor cells. Through exvivo gene therapy protocols tumor cells are engineered to express anαGal epitope. The cells are then killed (by gamma or ultravioletirradiation, heat, formaldehyde and the like) and administered to apatient. The αGal epitope causes opsonization of the tumor cell whichenhances tumor specific antigen presentation of antigens present in theentire tumor cell. An important feature of the invention contemplatesthe use of whole cells in the pharmaceutical compositions of theinvention. This provides for processing of tumor associated antigenspresent within the entire tumor cell regardless of whether thoseproteins have been affected by the addition of αGal epitopes or not.Since αGal modifications affect multiple glycoproteins and glycolipidson the cell surface, the animal's immune system will have an increasedopportunity to detect, process and generate antibodies and a cellularimmune response to tumor specific antigens. The animal's immune systemthus is stimulated to produce tumor specific antibodies and immunecells, which will attack and kill αGal negative tumor cells present inthe animal that bear tumor associated antigens which are common with theones provided by the engineered whole cell vaccine.

According to the invention a pharmaceutical composition is generated byintroducing a polynucleotide sequence, which encodes upon expression themurine αGT to whole tumor cells ex vivo. The recipient tumor cells maybe syngenic, allogenic, or autologous. The sequence is introducedthrough any nucleotide transfer vehicle which can comprise a viral ornon-viral vector, a plasmid or vector producer cells which produceactive viral particles. These gene transfer vehicles transform the tumorcells, and cause expression of foreign genetic material insertedtherein. The resulting gene product catalyzes the synthesis of αGalepitope, on cell surface glycoproteins and glycolipids present on saidcells. The invention contemplates the use of whole tumor cells withmultiple cell surface glycoproteins to maximize the binding of αGalepitopes by pre-existing anti-αGal antibodies thus enhancing binding ofthis complexes to the Fe receptors present on antigen presenting cellsand thus triggering antigen presentation of a plurality of tumorassociated antigens present in said vaccine tumor cell. In a morepreferred embodiment multiple types of transformed cells may beadministered from the same tissue type, or cancer type thus furtherincreasing the number of different epitopes provided to increase theprobability of complete amelioration of tumor cells present in theindividual.

The invention comprises a pharmaceutical composition and a method formaking the same which includes a therapeutically effective amount of amixture of attenuated tumor cells said mixture comprising a plurality ofcell surface glycoproteins which include an αGal epitope and a carrier.In a preferred embodiment the cells are whole cells. Methods for makingthe compositions include obtaining a collection of live tumor cells,transforming said cells with a nucleotide sequence that encodes uponexpression a an αGT so that an αGal epitope is presented on cell surfaceglycoproteins of said cells. The cells are then killed and combined witha pharmaceutical carrier for administration.

DEFINITIONS

Various terms relating to the compositions and methods of the presentinvention are used herein above and also throughout the specificationand claims.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range and include each integer within thedefined range. Amino acids may be referred to herein by either theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes. Unless otherwise provided for, software,electrical, and electronics terms as used herein are as defined in TheNew IEEE Standard Dictionary of Electrical and Electronics Terms (5^(th)edition, 1993). The terms defined below are more fully defined byreference to the specification as a whole.

The term “α-(1,3) Galactosyl Transferase encoding sequence”, or “αGTencoding sequence” is meant any polynucleotide sequence which encodes aprotein that forms α-galactosyl (αGal) epitopes by the followingreaction:

Galβ(1,4)G1cNAc-R UDP-Gal→Galα(1-3)Galβ(1,4)GlcNAc-R

This can include variants, modifications, truncations and the like aswell as murine sequences, bovine or sequences from any other sourceknown to those of skill in the art and available in Genbank, otherpublications or databases which retain the function of theaforementioned reaction.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerise chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “animal” as used herein should be construed to include allanti-αGal synthesizing animals including those which are not yet knownto synthesize anti-αGal. For example, some animals such as those of theavian species, are known not to synthesize αGal epitopes. Due to theunique reciprocal relationship among animals which synthesize eitheranti-αGal or αGal epitopes, it is believed that many animals heretoforeuntested in which αGal epitopes are absent may prove to be anti-αGalsynthesizing animals. The invention also encompasses these animals.

The term “antibody” includes reference to antigen binding forms ofantibodies (e.g., Fab, F(ab)2). The term “antibody” frequently refers toa polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments such as single chain Fv, chimeric antibodies (i.e.,comprising constant and variable regions from different species),humanized antibodies (i.e., comprising a complementarity determiningregion (CDR) from a non-human source) and heteroconjugate antibodies(e.g., bispecific antibodies).

The term “anti-αGal” includes any type or subtype of immunoglobulinrecognizing the αGal epitope, such as IgG, IgA, IgE or IgM anti-αGalantibody.

As used herein, the term “antigen” is meant any biological molecule(proteins, peptides, lipids, glycans, glycoproteins, glycolipids, etc)that is capable of eliciting an immune response against itself orportions thereof, including but not limited to, tumor associatedantigens and viral, bacterial, parasitic and fungal antigens.

As used herein, the term “antigen presentation” is meant the biologicalmechanism by which macrophages, clendritic cells, B cells and othertypes of antigen presenting cells process internal or external antigensinto subfragments of those molecules and present them complexed withclass I or class II major histocompatibility complex or CD1 molecules onthe surface of the cell. This process leads to growth stimulation ofother types of cells of the immune system (such as CD4+, CD8+, B and NKcells), which are able to specifically recognize those complexes andmediate an immune response against those antigens or cells displayingthose antigens.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences and is intended to be included whenever areference to a specific sequence is made. With respect to particularnucleic acid sequences, conservatively modified variants refer to thosenucleic acids which encode identical or conservatively modified variantsof the amino acid sequences. Because of the degeneracy of the geneticcode, a large number of functionally identical nucleic acids encode anygiven protein. For instance, the codons GCA, GCC, GCG and G-CU allencode the amino acid alanine. Thus, at every position where an alanineis specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid. One of ordinary skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine; andUGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W.H. Freeman and Company.

We define the “percentage of sequence identity” of two aminoacidsequences as the number of identical aminoacids shared by these twoaminoacid sequences after a pairwise alignment divided by the totallength of the shortest sequence of the pair.

We define the “percentage of sequence similarity” of two aminoacidsequences as the number of identical aminoacids plus conservativeaminoacid substitutions shared by these two sequences after a pairwisealignment, divided by the total length of the shortest sequence of thepair.

By “encoding” or “encoded”, “encodes”, with respect to a specifiednucleic acid, is meant comprising the information for translation intothe specified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed.

With respect to proteins or peptides, the term “isolated protein (orpeptide)” or “isolated and purified protein (or peptide)” is sometimesused herein. This term may refer to a protein that has been sufficientlyseparated from other proteins with which it would naturally beassociated, so as to exist in “substantially pure” form. Alternatively,this term may refer to a protein produced by expression of an isolatednucleic acid molecule.

With reference to nucleic acid molecules, the term “isolated nucleicacid” is sometimes used. This term, when applied to DNA, refers to a DNAmolecule that is separated from sequences with which it is immediatelycontiguous (in the 5′ and 3′ directions) in the naturally occurringgenome of the organism from which it was derived. For example, the“isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eukaryote. An “isolated nucleic acidmolecule” may also comprise a cDNA molecule. With respect to RNAmolecules, the term “isolated nucleic acid” primarily refers to an RNAmolecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from RNA molecules with which it would beassociated in its natural state (i.e., in cells or tissues), such thatit exists in a “substantially pure” form (the term “substantially pure”is defined below).

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transduction” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected MRNA).

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes of that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

The term “opsonization” of an antigen or a tumor cell is meant bindingof the anti-αGal epitopes present in the antigen or on the surface of atumor cell by anti-αGal antibodies thereby enhancing phagocytosis of theopsonized antigen or tumor cell by macrophages, dendritic cells, B cellsor other types of antigen presenting cells through binding of the Fcportion of the antibodies to the Fc receptors present on the surface ofantigen presenting cells.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons as “polynucleotides” as thatterm is intended herein.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein, butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, phosphorylation,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “substantially the same” refers to nucleic acid or amino acidsequences having sequence variation that do not materially affect thenature of the protein (i.e. the structure, stability characteristics,substrate specificity and/or biological activity of the protein). Withparticular reference to nucleic acid sequences, the term “substantiallythe same” is intended to refer to the coding region and to conservedsequences governing expression, and refers primarily to degeneratecodons encoding the same amino acid, or alternate codons encodingconservative substitute amino acids in the encoded polypeptide. Withreference to amino acid sequences, the term “substantially the same”refers generally to conservative substitutions and/or variations inregions of the polypeptide not involved in determination of structure orfunction.

With respect to antibodies, the term “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein ofinterest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

A “coding sequence” or “coding region” refers to a nucleic acid moleculehaving sequence information necessary to produce a gene product, whenthe sequence is expressed.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. This same definition is sometimes applied to the arrangementother transcription control elements (e.g. enhancers) in an expressionvector.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

The terms “promoter”, “promoter region” or “promoter sequence” refergenerally to transcriptional regulatory regions of a gene, which may befound at the 5′ or 3′ side of the coding region, or within the codingregion, or within introns. Typically, a promoter is a DNA regulatoryregion. capable of binding RNA polymerase in a cell and initiatingtranscription of a downstream (3′ direction) coding sequence. Thetypical 5′ promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence is a transcription initiation site (conveniently defined bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

The term “therapeutically effective amount” is meant an amount oftreatment composition sufficient to elicit a measurable decrease in thenumber, quality or replication of previously existing tumor cells asmeasurable by techniques including but not limited to those describedherein.

The term “tumor cell.” is meant a cell which is a component of a tumorin an animal, or a cell which is determined to be destined to become acomponent of a tumor, i.e., a cell which is a component of aprecancerous lesion in an animal. Included within this definition aremalignant cells of the hematopoietic system which do not form solidtumors such as leukemias, lymphomas and myelomas.

The term “tumor” is defined as one or more tumor cells capable offorming an invasive mass that can progressively displace or destroynormal tissues.

The term “malignant tumor” are those tumors formed by tumor cells thatcan develop the property of dissemination beyond their original site ofoccurrence.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus towhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “nucleic acid construct” or “DNA construct” is sometimes usedto refer to a coding sequence or sequences operably linked toappropriate regulatory sequences and inserted into a vector fortransforming a cell. This term may be used interchangeably with the term“transforming DNA”. Such a nucleic acid construct may contain a codingsequence for a gene product of interest, along with a selectable markergene and/or a reporter gene.

The term “selectable marker gene” refers to a gene encoding a productthat, when expressed, confers a selectable phenotype such as antibioticresistance on a transformed cell.

The term “reporter gene” refers to a gene that encodes a product whichis easily detectable by standard methods, either directly or indirectly.

A cell has been “transformed” or “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which, the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

The term “treat” or “treating” with respect to tumor cells refers tostopping the progression of said cells, slowing down growth, inducingregression, or amelioration of symptoms associated with the presence ofsaid cells.

The term “xenogeneic cell” refers to a cell that derives from adifferent animal species than the animal species that becomes therecipient animal host in a transplantation or vaccination procedure.

The term “allogeneic cell” refers to a cell that is of the same animalspecies but genetically different in one or more genetic loci as theanimal that becomes the “recipient host”. This usually applies to cellstransplanted from one animal to another non-identical animal of the samespecies.

The term “syngeneic cell” refers to a cell which is of the same animalspecies and has the same genetic composition for most genotypic andphenotypic markers as the animal who becomes the recipient host of thatcell line in a transplantation or vaccination procedure. This usuallyapplies to cells transplanted from identical twins or may be applied tocells transplanted between highly inbred animals.

DESCRIPTION OF THE FIGURES

FIG. 1 is a depiction of the pLNC-KG plasmid which may be used accordingto the invention.

FIGS. 2A-2C, SEQ ID NO:1, is the sequence of the pLNC-KG plasmiddepicted in FIG. 1.

FIG. 3 is a schematic of induction of Anti-αGal antibodies inα(1,3)-galactosyl transferase knockout (αGT KO) mice by immunizationwith rabbit red blood cells. To induce anti-αGal antibodies (Ab), micewere injected intraperitoneally with 10⁸ rabbit red blood cells (RRBC)twice, two weeks apart. One week after the last immunization, bloodsamples were obtained and anti-αGal antibody titers were determined byELTSA. All mice used in this study developed high titer of anti-αGal Ab.

FIG. 4 is a schematic showing survival test after subcutaneous injectionof a lethal dose of non-irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanomacells. Females and males 8 to 14 weeks old H-2 b/b haplotype αGT KO micewere used in this study. All mice were immunized with RRBC as showed inFIG. 3. One week after the last immunization, mice received a lethalsubcutaneous (subcutaneous) challenge of 1×10⁵ of either of these threecell lines: a) wild type B16.BL6 melanoma cell line (αGal⁽⁻⁾), b) B16cell retrovirally transduced with a vector expressing Neo-R gene(B16.LNL, mock control αGal⁽⁻⁾) or c) B16 cells retrovirally transducedwith a vector expressing both, NeoR gene and αGT (B16 αGal⁽⁺⁾, pLNCKG).After challenge, tumors were measured in a blinded manner twice a week.

FIGS. 5 a and 5 b are graphs showing tumor size after subcutaneousinjection (5 a—15 days after challenge, 5 b—30 days after challenge) oflethal doses of non-irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanoma cells inαGT knockout mice. Palpable subcutaneous tumors were measured with acaliper in three perpendicular axes the volume calculated and expressedin mm³. The figure depicts tumor sizes at 15 and 30 days aftersubcutaneous injection with the respective B16 cell line described inFIG. 4. The bars represent the mean and errors bars the SEM.

FIG. 6 is a graph showing tumor growth kinetics after subcutaneousinjection of a lethal doses of non-irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16melanoma cells in αGT KO mice. Tumor sizes were measured as in FIG. 5 at15, 23 and 29 days after challenge with: wild type B16 (αGal⁽⁻⁾),B16.LNL (control αGal⁽⁻⁾ and αGal⁽⁺⁾ B16.

FIGS. 7 a and 7 b are graphs showing survival analysis of αGT KO micelethally injected with non-irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanomacells. Mice treated as described in FIG. 4 were studied for 90 days.Kaplan-Meier survival analysis and long-rank survival curves comparisonswere performed using statistics software.

FIG. 8 is a schematic depicting the experimental design for survivaltest after the lethal subcutaneous injection of non-irradiated αGal⁽⁻⁾B16 melanoma cells in mice that survived a lethal injection ofnon-irradiated αGal⁽⁺⁾ B16 melanoma cells.

FIG. 9 is a graph showing survival analysis of knockout mice. Mice thatsurvived the first challenge with αGal⁽⁺⁾ B16 cells from FIG. 7, weresubsequently challenged with a second subcutaneous dose of nativeαGal⁽⁻⁾ B16 (FIG. 8). Kaplan-Meier analysis was performed for a periodof 60 days after the injection of αGal⁽⁻⁾ B16. Naïve mice receivingαGal⁽⁻⁾ B16 subcutaneous were used as controls.

FIG. 10 is a schematic showing induction of melanoma specific cytotoxicT cells in mice that survived a lethal dose injection of non-irradiatedαGal⁽⁺⁾ cells. Splenocytes from two mice that survived the firstchallenge with αGal⁽⁺⁾ B16 cells from FIG. 7, were used to generateCytotoxic T Lymphocytes (CTL) in vitro. Splenocytes were harvested 90clays after the injection of αGal⁽⁺⁾ B16 from tumor free mice andmelanoma specific cultures were generated by culturing splenocytes withirradiated αGal⁽⁻⁾ B16 cells during 5 days in the absence of IL-2. CTLwere harvested and tested against the specific target αGal⁽⁻⁾ B16 andthe non-specific syngeneic cell lines colon carcinoma MC38 and T celllymphoma EL-4. Specific cytotoxicity was determined after 4 h ofincubation of CTL with specific and non-specific targets by measuringLDI-I release in the culture supernatant.

FIG. 11 is a graph showing the results of induction of B16melanoma-specific cytotoxic T cells in mice that survived a lethal doseinjection of non-irradiated αGal⁽⁺⁾ cells as depicted in FIG. 10.

FIG. 12 is a schematic showing the experimental design of disseminatedmelanoma metastasis model in the αGT knockout mice by intravenousinjection of a lethal close of non-irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16melanoma cells. Females and males αGT KO mice were immunized with RRBCto increase the anti-αGal Ab titers as in FIG. 3. One week after thelast immunization, mice were intravenously injected with αGal⁽⁻⁾ B16 orαGal⁽⁺⁾ B16 in the tail vein. Three weeks after the injection, lungswere harvested and lung melanoma metastases were enumerated.

FIG. 13 is a graph showing the statistical results of the experiment inFIG. 12.

FIG. 14 is a schematic showing the experimental design for prevention ofsubcutaneous αGal⁽⁻⁾ melanoma tumor development in αGT knockout miceafter vaccination with irradiated αGal⁽⁺⁾ B16 melanoma cells. Cellvaccines were prepared using the B16-derived cell lines described inprevious experiments, which are: native αGal⁽⁻⁾ B16, αGal⁽⁻⁾ B16.LNLtransduced with control vector and αGal⁽⁺⁾ B16 cells transduced with thevector encoding the murine αGT gene. Cell vaccines were prepared byγ-irradiation (250 Gy) to prevent cell proliferation and stored infreezing media until use. Before injection cell vaccines were thawed,washed counted and injected subcutaneously suspended in Hanks' BalancedSalt Solution (HBSS). All αGT KO mice used were injected with RRBC as inFIG. 3. One week after the last RRBC injection, mice received the firstclose of cell vaccine. Two weeks later the cell vaccination wasrepeated. The dose of each vaccine was 10⁶ cells per mouse administeredsubcutaneous. Two weeks after the last cell vaccines, mice were injectedsubcutaneous with 10⁵ non-irradiated native αGal⁽⁻⁾ B16 cells andobserved for tumor development twice a week during 90 days.

FIG. 15 is a graph depicting the Kaplan-Meier survival analysis of theexperiment described in FIG. 14.

FIG. 16 shows the results of FACS analysis of recognition of TNF-α.Detection of intracellular TNF-α by melanoma specific T cells induced inmice vaccinated with irradiated αGal⁽⁺⁾ B16 cells after in vitrorecognition of αGal⁽⁻⁾ B16 melanoma cells. Splenocytes from tumor freemice were harvested from mice vaccinated with αGal⁽⁺⁾ B16 vaccines 90days after the injection of αGal⁽⁻⁾ B16 melanoma (mice that survivedfrom FIG. 15). To measure in vitro recognition of αGal⁽⁻⁾ B16intracellular TNF-α was detected by FACS. T cells were cultured for 6 hin presence or absence of stimulation with Brefeldin A to blocksecretion of cytokines. For maximum activation PMA/Ca⁺⁺ Ionophore wasused. Cells were cultured with 10⁵ irradiated B16 to measure specificrecognition or with 10⁵ CA320M as non-melanoma syngeneic negativecontrol cell line. After incubation cells were harvested, permeabilizedfixed and stained for intracellular TNF-α using PE-labeled anti-TNF-αmonoclonal Ab. Cells were analyzed using Coulter Flow cytometer,acquiring at least 10,000 gated lymphocytes.

FIG. 17 is a schematic showing the experimental design for therapeutictreatment of pre-established subcutaneous tumors through vaccinationwith irradiated melanoma cells. Therapeutic treatment of pre-establishedsubcutaneous αGal⁽⁻⁾ melanoma tumors through vaccination with irradiatedαGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanoma cells. Mice were injected with RRBC asexplained in FIG. 3. One week after the last RRBC injection, mice weresubcutaneously injected with 10⁵ non-irradiated αGal⁽⁻⁾ B16 melanomacells and randomized. In Experiment #1, two doses of cell vaccines wereadministered at 3 and 6 days after subcutaneous injection of αGal⁽⁻⁾B16. In Experiment #2 mice received three doses of the irradiated cellvaccines at 4, 11 and 18 days after the subcutaneous injection with liveαGal⁽⁻⁾ B16 cells. In these experiments, two cell vaccines were used:irradiated αGal⁽⁻⁾ B16 transduced with control vector or irradiatedαGal⁽⁺⁾ B16 cells.

FIGS. 18 a and 18 b show graphs of results of two different experimentsof tumor growth kinetics in mice with pre-established subcutaneousαGal⁽⁻⁾ melanoma tumors receiving vaccination with irradiated αGal⁽⁺⁾ orαGal⁽⁻⁾ B16 melanoma cells. Tumor sizes were measured at early timepoints in mice receiving or not cell vaccines. The values represent themean of tumor size of all mice in each group.

FIG. 19 is a graph showing Kaplan-Meier survival analysis of mice withpre-established subcutaneous αGal⁽⁻⁾ B16 melanoma tumors that receivedtherapeutic vaccination with irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanomacells. Mice injected with αGal⁽⁻⁾ B16 and treated or not with αGal⁽⁻⁾ orαGal⁽⁻⁾ B16 melanoma vaccines were evaluated for survival during 75 days(EXP #1).

FIG. 20 is a graph showing Kaplan-Meier survival analysis of mice withpre-established subcutaneous αGal⁽⁻⁾ B16 melanoma tumors that receivedtherapeutic vaccination with irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanomacells. Mice injected with αGal⁽⁻⁾ B16 and treated or not with αGal⁽¹⁾ orαGal⁽⁻⁾ B16 melanoma vaccines were evaluated for survival during 70 days(EXP#2).

FIG. 21 is a diagram showing the experimental design for therapeutictreatment of pre-established lung metastatic tumors. Treatment ofpre-established disseminated lung metastatic αGal⁽⁻⁾ melanoma tumorsthrough vaccination with irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 melanomacells. Mice were injected with RRBC as explained in FIG. 3. One weekafter the last RRBC injection, mice were intravenously (i.v.) injectedwith non-irradiated αGal⁽⁻⁾ B16 melanoma cells and randomized. They weresubsequently vaccinated with either αGal⁽⁻⁾ B16 or αGal⁽⁺⁾ B16 vaccinecells at days 4, 11 and 21 after establishment of the disseminatedmetastases. At day 30 animals were sacrificed and their lung tumorburden was evaluated.

FIG. 22 is a graph showing tumor burden in experiment 1 of the protocolshown in FIG. 21. In experiment #1 mice received 10⁵ αGal⁽⁻⁾ B16melanoma cells. Thirty days after the i.v injection of non-irradiatedmelanoma cells, mice were sacrificed and the lung melanoma metastaseswere enumerated.

FIG. 23 is a diagram showing a second experimental design fortherapeutic treatment of pre-established disseminated metastaticmelanoma tumors. In experiment #2 mice received 5×10⁵ αGal⁽⁻⁾ B16melanoma cells. After this, mice were treated subcutaneous with melanomacell vaccines. They received three doses of 2×10⁵ irradiated αGal⁽⁻⁾ B16transduced with control vector or irradiated αGal⁽⁺⁾ B16 cells on days4, 11 and 21 after the i.v injection of non-irradiated αGal⁽⁻⁾ B16.

FIGS. 24 a and 24 b are graphs showing the results of the experimentdescribed in FIG. 23 expressed as average weight of the lungs (24A) oras the mean lung tumor burden (24B).

FIG. 25 is a diagram showing the experimental design to demonstrate invitro induction of T cell immunity specific for αGal⁽⁻⁾ B16 melanomaafter vaccination with αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 cells. Mice were injectedwith RRBC as in FIG. 3. Two weeks after the last RRBC injection micereceived three subcutaneous injections of 2×10⁵ irradiated αGal⁽⁻⁾ B16cells transduced with control vector or irradiated αGal⁽⁺⁾ B16 cellvaccines. Two weeks after the last cell vaccine, splenocytes wereharvested and T cell studies were performed. To determine specificrecognition of αGal⁽⁻⁾ B16, intracellular TNF-α and up-regulation ofactivation markers CD25 and CD69 were measured.

FIG. 26 is a graph showing the specific induction of TNF-α in T cellprecursors of vaccinated animals that specifically recognize B16antigens (FIG. 25). To detect TNF-α T cells were cultured for 6 h inpresence or absence of stimulation with Brefeldin A to block secretionof cytokines. For maximum stimulation. PMA/Ca⁺⁺ Ionophore was used aspositive control. Cells were cultured with αGal⁽⁻⁾ B16 to measurespecific recognition or with CA320M, a non-melanoma syngeneic (H-2 b/b)small intestine cell line as negative control. After incubation cellswere harvested, and stained for intracellular TNF-α. Positive cells weredetected by FACS gating in lymphocytes in the Forward Scatter plot afteracquisition of at least 10,000 gated events. The plot depicts the MeanFluorescence Intensity (MFI) of TNF-α⁽⁺⁾ cells.

FIGS. 27 a and 27 b are graphs showing up-regulation of activationmarkers CD25 and CD69 in T cell precursors from animals vaccinated withαGal⁽⁺⁾ cells, that specifically recognize B16 antigens. Measurementswere performed after one day of culture under similar conditions asdescribed in FIG. 26, in absence of Brefeldin A. After incubation cellswere harvested and stained with PE-labeled monoclonal Ab anti-CD25 oranti-CD69. Acquisition was performed using Coulter Flow cytometer. Theplots depict percentage of positive CD25 or CD69 cells.

FIG. 28 shows a schematic of an In vivo demonstration of specific T cellmediated immunity with therapeutic effect against pre-establisheddisseminated metastasis of αGal⁽⁻⁾ B16 melanoma by adoptive T celltransfer from donor mice vaccinated with irradiated αGal⁽⁺⁾ or αGal⁽⁺⁾B16 cells. Donor mice were injected with RRBC as in FIG. 3. Two weeksafter the last RRBC injection mice received three subcutaneous injectionof 2×10⁵ irradiated αGal⁽⁻⁾ B16 transduced with control vector orirradiated αGal⁽⁺⁾ B16 cells vaccines. Two weeks after the last cellvaccine, splenocytes were harvested and transferred to sex-matchedrecipients. Four days previous to cell transfer, recipients wereinjected i.v. with non-irradiated αGal⁽⁻⁾ B16 to establish the lungmelanoma metastasis and randomized. Thirty days after T cell transfer,recipients were euthanized and lung melanoma metastasis burdendetermined by weighting the lungs and by enumerating melanoma tumors.

FIGS. 29 a and 29 b are graphs which depict the results of theexperiment described in FIG. 28. Bars represent the mean and error bars,the SEM. Two independent experiments were performed and they are shown.

FIG. 30 is a graph showing survival analysis of αGT KO mice vaccinatedwith αGal⁽⁺⁾ or αGal⁽⁻⁾ irradiated CA320M sarcoma cells aftersubcutaneous injection of a lethal close of non-irradiated αGal⁽⁻⁾CA320M sarcoma cells. Mice were injected with RRBC as in FIG. 3. Twoweeks after the last RRBC injection they were vaccinated subcutaneouswith either 1×10³ CA320M transduced with 10 MOI HDKgalΔsalI [αGal⁽⁻⁾vector] or FIDKgal1 [αGal⁽⁺⁾ vector] followed by 25 Gy irradiation.Twenty-one days later mice were injected subcutaneous with 1×10⁷non-irradiated CA320M cells. Null consisted of no vaccine. Survivalanalysis was performed during a period of observation of 60 days.

DETAILED DESCRIPTION OF THE INVENTION

The basic rationale for immune therapy against tumors is the inductionof an effective immune response against tumor-associated antigens (TAA),which in turn results in immune-mediated destruction of proliferatingtumor cells expressing these antigens. For an immune response to beeffective against TAAs comprising protein, these antigens must first beendocytosed by antigen presenting cells (APC) such as macrophages,dendritic cells and B cells. Within APCs, TAAs are degraded in thelysosomal compartment and the resulting peptides are expressed on thesurface of the macrophage cell membrane mostly in association with MHCClass II molecules but also in association with MHC class I molecules.This expression mediates recognition by specific CD4⁺ helper T cells andsubsequent activation of these cells to effect the immune response(Stevenson, 1991, FASEB J. 5:2250; Lanzavecchia, 1993, Science 260:937;Pardoll, 1993, Immunol. Today 14:310). The majority of human TAAmolecules have not been defined in molecular terms, preventing these foruse as targets for drug therapy or as anti-tumor vaccines.

The use of αGal epitopes in inducing a tumor specific response in aprophylactic vaccination-type of protocol has been proposed by others inthe field and has been shown to generate protection against a laterchallenge by the same tumor cells. There is no guarantee, however thatthe specific TAA's upon which the preventive immune response wasgenerated will be effective against subsequently developed tumor cells.The particular epitope of a TAA for which a prophylactic immune responseis generated through preventative vaccination could be specific to thattumor only, that type of cancer only, that patient only or that tissueonly etc. The identification of a universal TAA expressed by all cancershas remained elusive and that has prevented the widespread use ofprophylactic vaccination strategies for cancer prevention.

Instead of a general prophylactic approach, immune treatment of anindividual with recurring, or a diagnosed tumor would provide a moredirectly targeted method, however there is no consistent expectationthat a vaccine methodology will be efficacious as a treatment foralready present tumor cells. Several reports have shown effectiveness inprophylactic models, however with the same treatments showing lower orno effectiveness in therapeutic approaches. For example, preventivevaccination with recombinant Vaccinia virus encoding mouse TRP-1 wassuccessful in protecting against subsequent subcutaneous challenge withlive tumor B16 cells. This treatment was only partially effective in theprevention of lung melanoma metastasis and was not effective in thetreatment of pre-established subcutaneous melanoma, [Overwijk et al,Proc. Natl, Acad. Sci. USA (1999) 96: 2982-2987].

In a similar study, using synthetic oligodeoxynucleotide (ODN) adjuvantcontaining unmethylated cytosine-guanine motifs (CpG-ODN) and CTLA-4blockade, the effectiveness of peptide vaccines was evaluated inprophylactic, therapeutic and combination of both treatments. Theconclusion of the study was that neither treatment in the preventive orprophylactic mode was effective for B16 melanoma. The authorsdemonstrated that effective treatment and prolonged survival of mice wasobserved when mice were vaccinated, challenged with B16 and subsequentlythey received an additional therapeutic dose of the vaccine. In thisstudy however, vaccination was only partially effective for thetreatment of pre-established disease since all mice succumb of melanomain both the prophylactic, and therapeutic approach [Davila, Kennedy andCelis, Cancer Research (2003), 63: 3281-3288].

Many other studies have focused only on the preventive treatment ofsubcutaneous melanoma but only few of these have demonstrated asignificant and effective therapeutic effect on tumor growth rate butthough a reduction in tumor growth rate was not reflected in animalsurvival [Wang et al, Cancer Research (2003) 63: 2553-2560; CurrentProtocols of Immunology, Chapter 20, “B16 as a Model for HumanMelanoma”].

To further complicate the issue, the presence of tumor specificlymphocytes has been shown to be insufficient to induce tumordestruction. In fact, it has been demonstrated recently that thepresence of large amounts of tumor-specific T cells was not sufficientto prolong the survival of tumor-bearing mice [Overwijk et al, Journalof Experimental Medicine, (2003) 198: 569-580]. The authors demonstratedthat T cell stimulation through antigen-specific vaccination with analtered peptide ligand, rather than the native self-peptide, andco-administration of a T cell growth and activation factor were threeelements strictly necessary to induce tumor regression.

Most tumor cells have unique expression profiles of TAA, but in manycases these TAA are unknown or very difficult to determine or isolatefor individual tumors. Moreover, for most tumors that escape immunesurveillance the immune system does not recognize these TAA as foreignantigens because either they are not presented in the context of acellular “danger” signal or because the immune system has been tolerizedto those antigens and recognizes them as “self” antigens. The use ofwhole cell vaccines alleviates the first difficulty, as it provides awhole repertoire of TAA without the need to isolate or characterizethose antigens. The use of allogeneic whole cell vaccines provides TAAwhich might present allelic differences among individuals and thereforemight break the tolerance of the immune system to those TAA. Wholecancer vaccines have been also genetically modified to express moleculesthat enhance the immune response such as GM-CSF [Dranoff et al Proc.Natl. Acad. Sci. USA (1993) 90: 3539]. Genetically modified orunmodified whole cell allogeneic cancer vaccines are showing anti-tumoractivity and survival benefits in clinical trials, thereby validatingthe hypothesis that immune rejection of laboratory produced human cancercell lines can induce destruction of patient's malignancies. Thesevaccines function by direct stimulation of cellular immune effectors bydirect presentation of TAA in the context of the tumor vaccine Class IMHC which leads to direct activation of cytotoxic T lymphocytes andnatural killer cells [van Elsas et al. J. Exp. Medicine (1999) 190:355-366]. Stimulation of antitumor responses using the previouslymentioned approaches has been frequently associated with the triggeringof autoimmune disease against the normal tissue of the same cellulartype as the tumor tissue. Moreover, these vaccines do not exploit themechanisms of the humoral branch of the immune system to recognize TAAand to enhance antigen presentation and complement mediated destructionof tumor cells. There are several reasons why a strong humoral immuneresponse should produce a more effective anti-tumor response: I)antibodies that opsonize vaccine cells by binding to specific antigenson the cell surface would promote their phagocytosis by macrophages bybinding of the Fc portion of the antibodies to the Fc receptors presenton antigen presenting cells. II) Fc receptor targeting accomplishesseveral important functions for effective vaccine performance including:promoting the efficient uptake of antigen for both MHC Class I and IIantigenic presentation; promoting APC activation and promoting thematuration of dendritic cells. III) The uptake of opsonized cells byantigen presenting cells via Fc receptor mediated endocytosis may becritical to generating an effective anti-tumor CTL response, since itpromotes the activation of MHC class I restricted responses by CD8⁺T-cells through a cross-presentation pathway. IV) Vaccines that cannotstimulate a humoral immune response are limited in their ability toinduce cellular immunity by HLA restriction. CTLs are HLA restricted,and will only destroy the vaccine cells that present tumor antigens onself-class I MHC molecules. NK cells will destroy the tumor vaccinecells if the MHC interaction is poor producing a poor immune response.V) The signals that activate APCs come either directly or indirectlyfrom the naturally acquired immune response. The APCs that ingest tumorvaccine cells must be activated before they can effectively presentantigen. Moreover, presenting antigens to immature APCs, without therequired activation signals, can suppress the immune response.Furthermore, activated APCs can activate CTLs which cannot kill withoutactivation, even if they recognize their cognate antigen on the HLAmatched tumor cells.

From the above discussion it is clear that innovative tumor vaccines areneeded in the tumor vaccine field, where the specific needs are: I) todevelop tumor vaccines which can cause regression of pre-existing anddisseminated tumors or at least that can slow down tumor growth rates intherapeutic settings without the need of preventive vaccination regimes,II) to develop vaccines that stimulate both the cellular and humoralbranches of the immune system, and III) to develop vaccines that do nothave secondary undesired effects such as the triggering of autoimmunedisease.

Applicants' invention provides therapeutic tumor vaccine formulationsthat satisfy those requirements. The invention comprises the use of genetransfer technique to engineer tumor cells to synthesizeα(1,3)-Galactosyl epitopes in vitro. The use of the cell's own machineryand the use of a mixture of a number of allogeneic or syngeneic tumorcells that have been so engineered provide for epitopes to be generatedon multiple cell surface glycoproteins and glycolipids to providemultiple opportunities for antibodies to be generated to TAAs onindividual or separate cells. The cells used in these vaccines areestimated to contain between one and two million αGal epitopes. Thislarge number of binding sites for naturally preexisting anti-αGal Abresults in a high density of opsonization followed by complementdestruction which sets off a variety of processes that activate both theImmoral and cellular branches of the immune system. The presence of sucha high density of αGal residues on the surface of allogeneic tumor cellsinduces a hyperimmune response analogous to xenograft hyperacuterejection at the site of the modified tumor cell injection. Furthermore,these cancer vaccines are polyvalent meaning that they present multipletumor antigen targets to the immune system. This will result in a moreefficient treatment in that several TAAs will be presented and in a morewidely effective treatment as with the increased number of TAAspresented it is more likely that there will be overlap in epitopes fromdifferent individual tumors. Opsonized cells are readily ingested byphagocytes providing a mechanism whereby most of the tumor antigens canbe simultaneously presented to the adaptive immune system. Within thesecells, proteins from the cancer vaccine cells will be digested and givenclass II MHC presentation thereby exposing the mutant proteins epitopesin the cancer cell to T-cell surveillance. In addition, the uptake ofopsonized cells by antigen presenting cells (APCs) via Fc receptormediated endocytosis may facilitate the activation of MHC class Irestricted responses by CD8⁺ cells through a cross presentation pathway.The immune system cascade set in motion by this process provides thestimulus to induce a specific T-cell response to destroy native tumorcells from an established human malignancy. Furthermore, theinflammatory environment induced by the primary immune response resultsin an amplification effect mediated by cytokines, histamines and otherup-regulated molecules that boost the T-cell response. T-cells activatedin this manner are directly capable of killing cancer cells. Animportant remark is that the addition of αGal epitopes to glycoproteinsand glycolipids present in the tumor vaccine will not restrict thedevelopment of an immune response only to those antigens that becomeglycosylated but to any antigen present within the tumor cell whether itis affected by glycosylation or not.

The effectiveness of this kind of whole cell vaccine at inducingtherapeutic tumor immunity has been verified in animal models usingknockout (KO) mice. KO mice were generated lacking a functional αGT geneto provide a small animal model to study the in-vivo immune responseagainst αGal epitopes on tumor cell lines. These mice can be immunizedwith rabbit red blood cells (rRBC) to stimulate a high titer ofanti-αGal Ab as found in human serum. Using these mice, applicants havedemonstrated that the immune system rejects αGal positive tumor cellsand that this rejection leads to the development of T-cell immunityextended to αGal negative tumor cells. This animal model was also usedto simulate a clinical application where αGal negative tumor cells weregiven to the animal prior to treatment with the vaccine to simulate apre-established human malignancy. By these type of experimentsapplicants have shown that cell-mediated immunity induced in miceimmune-treated with cells engineered according to the invention wereable to effectively treat subcutaneous and pulmonary pre-establishedlocal and disseminated tumors resulting not only in reduce tumor growthrates but more importantly in long term survival of treated mice.Adoptive cell transfer experiments showed conclusively thecell-dependent component in the rejection of pre-established tumors. Thetypical autoimmune depigmentation associated to whole cell tumorvaccination described using other approaches was never observed in micesubjected to this treatment highlighting the importance that differentimmune stimulation methods may have on the final outcome of the immuneresponse.

Thus applicants' invention relates to methods and compositions forcausing the selective targeting and killing of pre-established tumorcells. Through ex vivo gene therapy protocols tumor cells are engineeredto express a αGal epitopes. The cells are then irradiated or otherwisekilled and administered to a patient. The binding of αGal epitope bynaturally pre-existing anti-αGal antibodies causes opsonization of thetumor cells and enhances tumor specific antigen presentation. Animportant feature of the invention contemplates the use of whole cells,and a mixture of a plurality of transduced cells in the pharmaceuticalcompositions of the invention. Since αGal modifications affect multipleglycoproteins on the cell surface, the animal's immune system will havean increased opportunity to detect, process and generate antibodies totumor specific antigens.

According to the invention a pharmaceutical composition is generated byintroducing to tumor cells a polynucleotide sequence, which encodes uponexpression an αGT enzyme. The sequence is introduced through anynucleotide transfer vehicle, which can comprise a viral or non-viralvector. These gene transfer vehicles transform the tumor cells, andcause expression of foreign genetic material inserted therein. Theresulting gene product catalyzes the synthesis of an αGal epitope, oncell surface glycoproteins present on said cells. The inventioncontemplates the use of whole tumor cells with multiple cell surfaceglycoproteins to maximize the binding of αGal epitopes by pre-existinganti-αGal antibodies thus enhancing binding of this complexes to the Fcreceptors present on antigen presenting cells and thus triggeringantigen presentation of a plurality of tumor associated antigens presentin said vaccine tumor cell.

The invention also comprises a pharmaceutical composition and a methodfor making the same, which includes a therapeutically effective amountof a mixture of attenuated tumor cells said mixture comprising aplurality of cell surface glycoproteins which include αGal epitopes anda carrier. In a preferred embodiment the cells are whole cells. Methodsfor making the compositions include obtaining a collection of live tumorcells, transforming said cells with a nucleotide sequence that encodesupon expression an αGT so that an αGal epitope is presented on cellsurface glycoproteins of said cells. The cells are then killed,preferably by irradiation and combined with a pharmaceutical carrier foradministration.

Yet another embodiment of the invention comprises the transformation oftumor cells with a polynucleotide which will create an αGal epitope onthe tumor cells. One embodiment of the invention comprisestransformation of tumor cells with a nucleotide sequence which encodesupon expression, the enzyme α-(1,3)-galactosyl transferase (αGT). TheαGT cDNA has been cloned from bovine and murine cDNA libraries. Larson,R. D. et al. (1989) “Isolation of a cDNA Encoding Murine UDP galactose;βD-galactosyl-1,4-N Acetol-D-Glucosamine α1-3 Galactosyl Transferase:Expression Cloning by Gene Transfer”, PNAS, USA 86:8227; and Joziasse,D. H. et al., (1989) “Bovine α1-3 Galactosyl Transferase: Isolation andCharacterization of a cDNA Clone, Identification of Homologous Sequencesin Human Genomic DNA”, J. Biol Chem 264:14290. Any other nucleotidesequence which similarly will result in the tumor cells expressing anαGal epitope on the cell surface may be used according to the invention,for example other enzymes that catalyze this reaction or perhaps eventthe engineering of the cells to have additional glycoproteins present onthe cell surface hence the artificial creation of a TAA which can bepresented to the immune system.

The tumor cells used for the pharmaceutical composition of the inventionmay be autologous, or in a preferred embodiment may be allogeneic orsyngeneic. The transformed cells and the tumor cells to be treated musthave at least one epitope in common, but will preferable have many. Tothe extent that universal, or overlapping epitopes or TAA exist betweendifferent cancers, the pharmaceutical compositions may be quite widelyapplicable.

The invention also need not be limited to a cancer cell and can includeany cell surface glycoprotein-containing cell, or cell component thathas a site for an αGal epitope. This may include certain viruses,neoplastic cells, and the like.

The nucleic acid sequence that encodes the αGal epitope generatingprotein is contained in an appropriate expression vehicle, whichtransduces the tumor cells. Such expression vehicles include, but arenot limited to, eukaryotic vectors, prokaryotic vectors (such as, forexample, bacterial vectors), and viral vectors.

In one embodiment, the expression vector is a viral vector. Viralvectors which may be employed include, but are not limited to,retroviral vectors, adenovirus vectors, Herpes virus vectors, andadeno-associated virus vectors, or DNA conjugates.

In a preferred embodiment, a viral vector packaging cell line istransduced with a viral vector containing the nucleic acid sequenceencoding the agent which induces the destruction of the tumor cells byantibody binding and complement activation. The viral particles producedby the packaging cell line are harvested and used to transduce the tumorcells which will be administered as the anti-tumor vaccine.

Traditionally, the viral vector is a retroviral or adenoviral vector.Examples of retroviral vectors which may be employed include, but arenot limited to, Moloney Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus.

Retroviral vectors are useful as agents to mediate retroviral-mediatedgene transfer into eukaryotic cells. Retroviral vectors are generallyconstructed such that the majority of sequences coding for thestructural genes of the virus are deleted and replaced by the gene(s) ofinterest. Most often, the structural genes (i.e., gag, pol, and env),are removed from the retroviral backbone using genetic engineeringtechniques known in the art.

These new genes have been incorporated into the proviral backbone inseveral general ways. The most straightforward constructions are ones inwhich the structural genes of the retrovirus are replaced by a singlegene which then is transcribed under the control of the viral regulatorysequences within the long terminal repeat (LTR). Retroviral vectors havealso been constructed which can introduce more than one gene into targetcells. Usually, in such vectors one gene is under the regulatory controlof the viral LTR, while the second gene is expressed either off aspliced message or is under the regulation of its own, internalpromoter.

Efforts have been directed at minimizing the viral component of theviral backbone, largely in an effort to reduce the chance forrecombination between the vector and the packaging-defective helpervirus within packaging cells. A packaging-defective helper virus isnecessary to provide the structural genes of a retrovirus, which havebeen deleted from the vector itself.

In one embodiment, the retroviral vector may be one of a series ofvectors described in Bender, et al., J. Virol. 61:1639-1649 (1987),based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650)containing a series of deletions and substitutions to reduce to anabsolute minimum the homology between the vector and packaging systems.These changes have also reduced the likelihood that viral proteins wouldbe expressed. In the first of these vectors, LNL-XHC, there was altered,by site-directed mutagenesis, the natural ATG start codon of gag to TAG,thereby eliminating unintended protein synthesis from that point.

In Moloney murine leukemia virus (MoMuLV), 5′ to the authentic gagstart, an open reading frame exists which permits expression of anotherglycosylated protein (pPr80.sup.gag). Moloney murine sarcoma virus(MoMuSV) has alterations in this 5′ region, including a frameshift andloss of glycosylation sites, which obviate potential expression of theamino terminus of pPr80.sup.gag. Therefore, the vector LNL6 was made,which incorporated both the altered ATG of LNL-XHC and the 5′ portion ofMoMuSV. The 5′ structure of the LN vector series thus eliminates thepossibility of expression of retroviral reading frames, with thesubsequent production of viral antigens in genetically transduced targetcells. In a final alteration to reduce overlap with packaging-defectivehelper virus, Miller has eliminated extra env sequences immediatelypreceding the 3′ LTR in the LN vector (Miller, et al., Biotechniques,7:980-990, 1989). One example of a vector which may be used according tothe invention is shown in FIG. 1, which the sequence shown in FIG. 2,SEQ ID NO:1.

The paramount need that must be satisfied by any gene transfer systemfor its application to gene therapy is safety. Safety is derived fromthe combination of vector genome structure together with the packagingsystem that is utilized for production of the infectious vector. Miller,et al. have developed the combination of the pPAM3 plasmid (thepackaging-defective helper genome) for expression of retroviralstructural proteins together with the LN vector series to make a vectorpackaging system where the generation of recombinant wild-typeretrovirus is reduced to a minimum through the elimination of nearly allsites of recombination between the vector genome and thepackaging-defective helper genome (i.e. LN with pPAM3).

In one embodiment, the retroviral vector may be a Moloney MurineLeukemia Virus of the LN series of vectors, such as those hereinabovementioned, and described further in Bender, et al. (1987) and Miller, etal. (1989). Such vectors have a portion of the packaging signal derivedfrom a mouse sarcoma virus, and a mutated gag initiation codon. The term“mutated” as used herein means that the gag initiation codon has beendeleted or altered such that the gag protein or fragment or truncationsthereof, are not expressed.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, and(3-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, TK, promoters,and B19 parvovirus promoters.

In another embodiment the invention comprises an inducible promoter. Onesuch promoter is the tetracycline-controlled transactivator(tTA)-responsive promoter (tet system), a prokaryotic inducible promotersystem which has been adapted for use in mammalian cells. The tet systemwas organized within a retroviral vector so that high levels ofconstitutively-produced tTA mRNA function not only for production of tTAprotein but also the decreased basal expression of the response unit byantisense inhibition. See, Paulus, W. et al., “Self-Contained,Tetracycline-Regulated Retroviral Vector System for Gene Delivery toMammalian Cells”, J of Virology, January. 1996, Vol. 70, No. 1, pp.62-67. The selection of a suitable promoter will be apparent to thoseskilled in the art from the teachings contained herein.

The vector then is employed to transduce a packaging cell tine to form aproducer cell line. Examples of packaging cells which may be transfectedinclude, but are not limited to the PE501, PA317, .psi.2, .psi.-AM,PAl2, T19-14×, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAM12,DAN and AMIZ cell lines. The vector containing the nucleic acid sequenceencoding the agent which is capable of providing for the destruction ofthe tumor cells upon expression of the nucleic acid sequence encodingthe agent, and activation of the complement cascade may transduce thepackaging cells through any means known in the art. Such means include,but are not limited to, electroporation, the use of liposomes, and CaPO₄precipitation.

In a preferred embodiment the invention comprises a viral vector whichcommonly infects humans and packaging cell line which is human based.For example vectors derived from viruses which commonly infect humanssuch as Herpes Virus, Epstein Barr Virus, may be used.

Tumors which may be treated in accordance with the present inventioninclude malignant and non-malignant tumors. Malignant (including primaryand metastatic) tumors which may be treated include, but are not limitedto, those occurring in the adrenal glands; bladder; bone; breast;cervix; endocrine glands (including thyroid glands, the pituitary gland,and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney;liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx;larynx; ovaries; penis; prostate; skin (including melanoma); testicles;thymus; and uterus. Examples of such tumors include apudoma, choristoma,branchioma, malignant carcinoid syndrome, carcinoid heart disease,carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, in situ, Krebs 2, Merkel cell, mucinous, non-small celllung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic,squamous cell, and transitional cell), plasmacytoma, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma,mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma,thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma,cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma,meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyonia,lymphangio sarcoma, pinealoma, carcinosarcoma, chondrosarcoma,cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma,leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma,ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing'sexperimental, Kaposi's, and mast-cell), neoplasms and for other suchcells.

Pharmaceutical Preparations

According to the invention attenuated αGal expressing tumor cells areused as either prophylactic or therapeutic vaccines to treat tumors.Thus the invention also includes pharmaceutical preparations for humansand animals involving these transgenic tumor cells (expressed as HAL HA2etc., see Table 1). Those skilled in the medical arts will readilyappreciate that the doses and schedules of pharmaceutical compositionwill vary depending on the age, health, sex, size and weight of thehuman and animal. These parameters can be determined for each system bywell-established procedures and analysis e.g., in phase I, II and IIIclinical trials and by review of the examples provided herein.

For administration, the attenuated tumor cells can be combined with apharmaceutically acceptable carrier such as a suitable liquid vehicle orexcipient and an optional auxiliary additive or additives. The liquidvehicles and excipients are conventional and are commercially available.Illustrative thereof are distilled water, physiological saline, aqueoussolutions of dextrose and the like.

Suitable formulations for parenteral, subcutaneous, intradermal,intramuscular, oral or intraperitoneal administration, include aqueoussolutions of active compounds in water-soluble or water-dispersibleform. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, include for example, sodium carboxymethylcellulose, sorbitol and/or dextran, optionally the suspension may alsocontain stabilizers. Also, tumor vaccine cells can be mixed with immuneadjuvants well known in the art such as Freund's complete adjuvant,inorganic salts such as zinc chloride, calcium phosphate, aluminumhydroxide, aluminum phosphate, saponins, polymers, lipids or lipidfractions (Lipid A, monophosphoryl lipid A), modified oligonucleotides,etc.

In addition to administration with conventional carriers, activeingredients may be administered by a variety of specialized deliverydrug techniques which are known to those of skill in the art. Thefollowing examples are given for illustrative purposes only and are inno way intended to limit the invention.

The invention will now be described with respect to the followingexamples; however, the scope of the present invention is not intended tobe limited thereby. All citations to patents and journal articles arehereby expressly incorporated by reference.

Example 1 Production of Retroviral Vector Expressing αGT, pLNCKG

A 1,077 bp fragment of murine αGT gene was PCR amplified by a forwardprimer, 5′-ACAAAAGCTTGACATGGATGTCAAGGGAAAAGTAAT-3′, which contains aKozak sequence to enhance the translation of αGT, and a reverse primer,5′-AATTATCGATTCAGACATTATTTCTAAC-3′, and then cloned into the ClaI andHindIII sites of pLNCX to produce pLNCKG retroviral vector (FIG. 1).This vector was transfected into the packaging cell line 293.AMIZ [Youngand Link “Chimeric retroviral helper virus and picornavirus IRESsequence to eliminate DNA methylation for improved retroviral packagingcells” J. Virol. (2000) 74: 5242-5249] to generate the vector producercell line 293.AMTZ/LNCKG. Transfected cells were selected in presence ofG418 and Zeocin™ for two weeks. Mixed population of selected cells wassubcloned by limiting dilutions. Single cell-derived VPC were screenedfor their ability to effectively transduce human epithelial cancer celllines established from different tissues. The clone which supernatantconsistently yielded highest transduction efficiency and αGT expressionon a panel of human epithelial cancer cell lines was identified anddesignated 293Z.CKG VPC. A master cell bank, working cell bank andproduction lot was generated for 293Z.CKG VPC was originated from onevial of the seed bank, expanded in flasks at 37° C.±1° C. in 5%±1% CO2.The culture medium was RPMI-1640 supplemented with 10% fetal bovineserum (FBS) and 2 mm L-glutamine. When the 293Z.CKG VPC reachedsufficient density, the culture fluids (supernatant) are harvested,filtered, and pooled into a sterile container. The pool is thoroughlymixed and then aseptically filled into labeled, sterile plastic bottles.(Labels contain the product name, lot number and date of filling.) Thefill bottles are frozen and stored at or below −60° C. Aliquots aresubmitted for safety testing. Retrovirus-containing supernatants from293Z.CKG VPC were used to transduce different human cancer cell lines(mentioned in examples below) to establish the αGal⁽⁺⁾ whole cellvaccines.

Example 2 Transduction of B16.BL6 Melanoma Cells with LNCKG RetroviralVector

To generate αGal⁽⁺⁾ B16 cells, 2×10⁶ cells were transduced with 2 mL ofsupernatant containing the LNCKG retrovirus with an infectious titer of2×10⁶ tu/mL. Cells were selected for resistance to Neomycin by atwo-week selection in medium supplemented with G418 1 mg/mL. After thisperiod of selection, cells were stained for expression of the αGalepitope with a chicken anti-αGal polyclonal antibody and sorted byfluorescence activated cell sorting.

Example 3 Induction of Anti-αGal Antibodies inα(1,3)-Galactosyltransferase Knockout (αGT KO) Mice by Immunization withRabbit Red Blood Cells

Females and males 8 to 14 weeks old α-(1,3)galactosyltransferase (αGT)knockout (KO) mice were used in this study. Mice were initially of mixedhaplotype (H-2 b/d) and by breeding and selection the current colony ofαGT KO mice was obtained consisting in F4 inbreeding generation of H-2b/b haplotype. These animals produce low titers of natural antibodiesagainst αGal epitopes. To increase the titer of anti-αGal Ab mice wereimmunized intraperitoneally (i.p.) with 1×10⁸ Rabbit Red Blood Cellstwice, two weeks apart. The titers of anti-αGal Ab were checked one weekafter the last RRBC injection to corroborate that all mice in the studyhave high titers of anti-αGal Ab. All mice used in this study have highanti-αGal Ab titers greater than 1:500 dilution, measured by ELISA. Arepresentative experiment is shown in FIG. 3.

Example 4 Survival Test After Subcutaneous Injection of a Lethal Dose ofNon-Irradiated αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 Melanoma Cells

The goal of this experiment was to determine if the expression of αGalepitopes in cancer cells would lead to their in vivo hyperacuterejection in hosts with pre-existing anti-αGal Ab. This RRBC-injectedαGT KO mice were challenged with non-irradiated 10⁵ B16 melanoma cellsexpressing or not the αGal epitopes and tumor development was measured(FIG. 4).

FIG. 5 shows tumor size 15 days after the challenge. As expected, 11 of19 mice that received native αGal⁽⁻⁾ B16 developed measurable tumors twoweeks after the challenge. Similarly 10 of 20 mice receiving the B16.LNLαGal⁽⁻⁾ B16 cells developed tumors (57% and 50%, respectively. Chisquare p>0.05, not significant). However, when mice received theαGal⁽⁺⁾B16 cells, significant fewer animals developed tumors. Only 5 outof 20 animals (25%) developed palpable tumors (Chi square, p=0.03significantly different from control). In addition, tumors wereconsiderable larger in animals challenged with the control and mocktransduced cells compared with tumor developed in mice receiving theαGal-expressing cells. Average sizes of 200 mm³ tumors were observed inboth controls groups with differences between groups not beingstatistically significant (F test p=0.38). However, a significantdifference was observed in the tumor size in the group receiving theB16.αGal⁽⁺⁾ cells. The mean size tumor of the test group was 36 mm³,which represents about 80% reduction in the tumor size compared withcontrol animals (F test p=0.002).

This result demonstrates that fewer animals developed smaller tumorswhen challenged with αGal-expressing B16 cells indicating that anti-αGalpre-sensitized mice are able to mediate more effective in vivo clearanceof αGal expressing cells compared with the native or mock transducedαGal negative B16 controls.

Comparable results were observed when tumors were measured 30 days afterthe challenge. Thirteen mice had tumors out of 17 mice in the group thatreceived B16 wild type (76%). Similarly 13 out of 18 mice developedlarge tumors in the group receiving cells transduced with mock vector(72%). However, only 50% of mice had measurable tumors in the groupreceiving the αGal-expressing cells. More importantly, a total of fouranimals, two in each of the control groups, had to be euthanized becauseof the development of large tumors. None of the animals that receivedthe αGal expressing cells died by thirty days after tumor implantation,indicating an increase in the survival of animal challenged with αGalexpressing cells.

FIG. 6 shows the kinetics of tumor development after challenge withcontrol, mock and αGal-expressing B16 cells. Tumors in both controlαGal⁽⁻⁾ groups grew extremely fast almost doubling the volume every 7days. On the contrary, tumors from mice challenged with αGal⁽⁺⁾ B16cells grew significantly slower at early time points (p=0.03). Thissuggests that immune mechanism(s) may restrain the growth of the tumorand help prolong the survival of tumor-bearing mice.

Long-Term Survival of Mice After Lethal Challenge with αGalf⁽⁺⁾ B16Cells.

Mice from groups that received control αGal⁽⁻⁾, mock transduced αGal⁽⁻⁾B16 and αGal⁽⁺⁾ B16 were observed weekly during 90 days (FIG. 7). Asshown in the figure, none out of 10 animals receiving native B16survived the challenge and only one mice out of 11 receivingmock-transduced αGal⁽⁻⁾ B16 cells survived the subcutaneous injection.Both control groups showed very similar survival curves, indicating thatthe NeoR gene product is not inducing a significant change in therejection and/or immunogenicity of B16 (Logrank. Test p=0.5, differencesnot statistically significant). On the contrary, 47% of the animalsreceiving αGal-expressing non-irradiated B16 cells survived the lethalchallenge. Nine out of 19 mice remained tumor free for 80 days after thesubcutaneous injection. Using Kaplan-Meier survival analysis asignificant increase in the survival proportion was observed in thegroup subcutaneous injected with αGal⁽⁺⁾ B16 cells compared to controlgroups (Logrank Test p<0.02).

In both control groups injected with αGal⁽⁻⁾ B16, near 60% of theanimals died in the first 30 days after challenge. On the contrary theexperimental group injected with αGal⁽⁺⁾ B16 only five animals (26%) hadto be euthanized in the first month after challenge.

In a second independent experiment similar results were obtained. Only 2out of 19 mice injected subcutaneous with native αGal⁽⁻⁾ B16 survived.Comparably, only 4 out of 21 mice survived the lethal subcutaneouschallenge with mock transduced αGal⁽⁻⁾ B16 cells. On the contrary, 8 outof 20 mice injected subcutaneous with αGal⁽⁺⁾ B16 survived and remainedtumor free for more than 80 days.

Altogether this result demonstrates that the expression of αGT gene andthe change in the glycosilation pattern of B16 induce in vivodestruction of living cancer cells that helped reduce tumor growthprolonging the survival of tumor-bearing mice. More importantly, sinceabout 40% of challenged mice remained tumor free, the injection of αGalexpressing cells may lead to the induction of a strong immune responseable to control the growth of highly lethal αGal⁽⁻⁾ melanoma cells.

Survival After Lethal Challenge with Non-Irradiated αGal⁽⁺⁾ B16 CellsLead to Induction of Memory Protective Immunity Against Wild TypeαGal⁽⁻⁾ B16

We hypothesized that surviving animals from the subcutaneous injectionof non-irradiated αGal⁽⁺⁾ B16 cells, developed cellular immunityextended to the native αGal⁽⁻⁾ B16 tumor. To test this hypothesis allsurviving tumor-free mice were re-challenged with wild type B16 (αGalnegative). Aged-matched mice were included as controls and were alsoinjected with αGal⁽⁻⁾ B16 (FIG. 8). As expected 11 out of 12 controlmice challenged with αGal⁽⁻⁾ B16 died from subcutaneous melanomadeveloping large and pigmented tumors (FIG. 9). On the other hand, noneof the mice that survived the first encounter with non-irradiatedαGal⁽⁺⁾ B16 melanoma developed tumors. All 8 mice remained tumor freefor 70 days increasing significantly the survival of mice that firstrejected αGal⁽⁺⁾ B16 cells (logrank test p<0.001). Interestingly,protection against B16 melanoma was not associated with autoimmunedepigmentation (vitiligo) as previously described by others [Overwijk etal. “Vaccination with a recombinant vaccinia virus encoding a selfantigen induces autoimmune vitiligo and tumor cell destruction in mice:requirement for CD4+ T lymphocytes” Proc. Natl. Acad. Sci. USA (1999)96: 2982-2987; Overwijk et al. “Tumor regression and autoimmunity afterreversal of functionally tolerant state of self-reactive CD8+ T cells”J. Exp. Med. (2003) 198: 569-580; van Elsas et al. “Combinationimmunotherapy of B16 melanoma using anti-CTLA-4 and GM-CSF-producingvaccines induces rejection of subcutaneous and metastatic tumorsaccompanied by autoimmune depigmentation” J. Exp. Med. (1999) 190:355-366].

This result demonstrated that mice that survived the lethal challengewith αGal⁽⁺⁾ cells developed strong immunity against the native αGal⁽⁻⁾tumor. This cellular and possibly humoral immune response protectedsurviving mice from a second lethal challenge with wild tumor indicatingthat the immune response has been extended to native αGal⁽⁻⁾ tumor B16in all protected mice.

To further demonstrate the hypothesis that T cell mediated immunity wasinduced in protected mice, melanoma specific T cell cultures weregenerated and tested against αGal⁽⁻⁾ B16 melanoma, and the non-specificcell lines EL-4 and MC38 (FIG. 10).

As shown in FIG. 11, strong CTL able to lyse B16 melanoma specificallywere induced in protected mice measured by LDH release in target cells.

Altogether, these results demonstrate that the injection of αGal⁽⁺⁾cells increased the survival in mice. Moreover, mice that rejectedαGal⁽⁺⁾ B16 cells and survived the initial lethal injection, were ableto develop strong immunity extended against the native αGal⁽⁻⁾ B16melanoma tumor. This indicates that memory T cell mediated immunity wasinduced after rejection of αGal⁽⁺⁾ B16 cells able to recognize αGal⁽⁻⁾B16 tumor protecting mice from a lethal tumor dose.

Example 5 Disseminated Melanoma Metastasis Model in the αGT KnockoutMice by Intravenous Injection of a Lethal Dose of Non-Irradiated αGal⁽⁻⁾or αGal⁽⁻⁾ B16 melanoma cells

To further demonstrate that mice with high titer of anti-αGal Ab areable to reject αGal⁽⁺⁾ B16 melanoma cells, the lung melanoma metastasismodel was used. Mice were intravenously (i.v) injected with 10⁵non-irradiated αGal⁽⁻⁾ or αGal⁽⁺⁾ B16 melanoma cells. Three weeks later,the lung melanoma metastases were enumerated (FIG. 12). Mice injectedwith αGal⁽⁻⁾ B16 cells have many lung metastasis. On the contrary miceinjected with αGal⁽⁺⁾ B16 cells have reduced lung burden (FIG. 13).Moreover two out of 5 mice were tumor free. This result indicates thatpre-existing anti-αGal Ab played a major role in the clearance ofαGal⁽⁺⁾ B16 cells.

Example 6 Prevention of Subcutaneous Tumor Development by ProphylacticVaccination with αGal⁽⁺⁾ Cells

The goal of this experiment was to determine whether the prevention ofsubcutaneous tumors could be accomplished by vaccination with αGal⁽⁺⁾vaccines as previously described [LaTemple et al. “Increasedimmunogenicity of tumor vaccines complexed with anti-αGal: studies inknockout mice for α1,3galatosyltransferase” Cancer Res. (1999)59:3417-3423]. Mice were immunized with RRBC as described previously.One week after the last RRBC injection mice received the first dose ofirradiated cell vaccine. Mice were injected either with irradiatednative αGal⁽⁻⁾ B16, αGal⁽⁻⁾ B16 transduced with control vector (pLNL,encoding for Neomycin Resistance Gene) or with αGal⁽⁺⁾ B16 (transducedwith vector encoding the Neomycin Resistance Gene and the αGT gene).Some mice did not receive irradiated cell vaccines. The cell vaccinationwas repeated two weeks later. Two weeks after the last vaccination micewere injected subcutaneous with 10⁵ non-irradiated αGal⁽⁻⁾ B16 cells(FIG. 14). Tumors were measured twice a week for 90 days. As shown inFIG. 15, zero out of 10 mice that did not received B16 cell vaccinessurvived the challenge and died before 50 days after challenge. Someprotection was observed in mice vaccinated with native αGal⁽⁻⁾ B16 andwith αGal⁽⁻⁾ B16/NeoR vaccines. Four 4 out of 14 and 5 out of 12 micesurvived the B16 challenge, respectively. There is not statisticaldifference between mice vaccinated with B16 and B16/NeoR which indicatesthat under this conditions NeoR gene product does not increaseimmunogenicity of B16 (p>0.2, Logrank test). On the contrary,significant more protection was observed when mice were vaccinated withαGal⁽⁺⁾ B16 cells since 12 out of 20 mice survived the challenge andremained tumor free for more than 90 days (p<0.001, ANOVA, p=0.08Logrank test). This result demonstrates that vaccination with irradiatedαGal⁽⁺⁾ cells prevents the development of subcutaneous melanoma tumorsin 60% of αGal⁽⁺⁾ B16 vaccinated mice and significantly prolonged thesurvival of mice challenged with αGal⁽⁻⁾ B16 melanoma.

We hypothesized that T cells able to recognize the native αGal⁽⁻⁾ B16cells were induced after vaccination with αGal⁽⁺⁾ B16 vaccine in miceprotected from the injection with live αGal⁽⁻⁾ B16. To test thishypothesis, splenocytes from mice vaccinated with αGal⁽⁺⁾ vaccines wereharvested and cultured for 6 h in presence or absence of stimulation.For maximum stimulation PMA/Ca⁺⁺ Ionophore was used. Cells were culturedwith 10⁵ irradiated B16 cells to measure specific recognition or withCA320M, a non-specific αGal⁽⁻⁾ cell line with identical H-2 b/bhaplotype. After incubation cells were harvested and stained forintracellular TNF-α. Detection was performed by FACS gating inlymphocytes in the Forward Scatter plot (FIG. 16). T cells harvestedfrom, αGal⁽⁺⁾ B16 vaccinated mice were efficiently activated by PMA/Ca⁺⁺Ionophore. The percentage of lymphocytes activated by this polyclonalactivator method is considered the maximum activation detected in thisexperiment. Resting (unstimulated) T cells and T cells stimulated withCA320M were not able to produce TNF-α, indicating that no T cellsprecursors were induced after B16-αGal⁽⁺⁾ vaccination able to recognizeantigens in CA320M. On the contrary, vaccination with B16-αGal⁽⁺⁾induced T cell precursor that specifically recognize B16-αGal⁽⁻⁾ invitro. This result suggests that these T cells induced after vaccinationwith αGal⁽⁺⁾ B16 maybe responsible for tumor prevention in about half ofαGal⁽⁺⁾ B16 treated mice.

Example 7 Treatment of Pre-Established Subcutaneous Melanoma Tumors byαGal⁽⁺⁾ Cell Vaccination

Since the vaccination with native αGal⁽⁻⁾ B16 and αGal⁽⁻⁾mock-transduced B16/NeoR resulted in similar data in the experimentsshown above the following experiments were performed with vaccinationusing irradiated αGal⁽⁻⁾ B16/NeoR alone as negative control to increasethe power of the statistical analysis.

The goal of the next experiment was to determine whether the treatmentof pre-established subcutaneous melanoma tumors could be accomplished byvaccination with αGal⁽⁺⁾ B16 vaccines. It is not obvious that theeffective preventive treatment will be also effective in the treatmentof pre-established tumors. In the particular case of B16 melanoma astumor model, several strategies have been proved effective in preventiveschedules of immunization and have had low or no impact in the treatmentof pre-established subcutaneous melanoma tumors. For example, thevaccination with recombinant vaccinia virus encoding mouse mTRP-1prevents the development of subcutaneous melanoma tumors and has noimpact in the treatment of pre-established subcutaneous tumors [Overwijket al. “Vaccination with a recombinant vaccinia virus encoding a selfantigen induces autoimmune vitiligo and tumor cell destruction in mice:requirement for CD4+ T lymphocytes” Proc. Natl. Acad. Sci. USA (1999)96: 2982-2987]. Similarly, peptide specific immunization leads to theinduction of strong T cell immunity but it is rarely effective for thetreatment against pre-established tumors (Davila et al. “Generation ofantitumor immunity by cytotoxic T lymphocyte epitope peptidevaccination, CpG oligodeoxynucleotide adjuvant and CTLA-4 blockade”Cancer Research (2003) 63:3281-3288]. Moreover, the sole presence oftumor specific T cell is a condition necessary but it is not sufficientto effectively induce tumor eradication.

We hypothesized that αGa⁽⁺⁾ vaccines will induce strong cell dependenttumor immunity able to reject pre-established αGal⁽⁻⁾ tumors. To testthis hypothesis we conducted experiments designed to treatpre-established tumors (FIG. 17).

Mice were injected subcutaneous with non-irradiated 10⁵ B16 cells andrandomized. Three days after challenge they were vaccinated subcutaneouswith irradiated αGal⁽⁺⁾ B16/NeoR or with irradiated αGal⁽⁺⁾ B16. Threedays later the vaccination with irradiated cell vaccines was repeated.The baseline control mice received subcutaneous injection withnon-irradiated B16 and they did not receive irradiated cell vaccinetreatment (No vaccine group). FIG. 18 a and b show the kinetics of tumorgrowth of non-vaccinated mice (n=11), mice vaccinated with αGal⁽⁻⁾B16/NeoR (n=24) and mice receiving αGal⁽⁺⁾ B16 vaccine (n=23). The datarepresents the mean and error bars, the SEM. Statistical analysisindicates a significant difference of the slopes, when comparing controlmice with mice receiving αGal⁽⁺⁾ B16 cell vaccines (p<0.009). Thisresult indicates that mice vaccinated with irradiated αGal⁽⁺⁾ B16vaccines developed smaller tumors that grew slower.

In Experiment #2, mice received and additional dose of cell vaccine.Mice receiving only the subcutaneous injection with non-irradiatedαGal⁽⁻⁾ B16 developed large tumors that grew very fast during the firstmonth of observation (n=15). Similarly, mice injected with controlαGal⁽⁻⁾ B16 vaccine developed large tumors growing very fast (n=29). Thestatistical comparison of the slopes of these two groups indicated thatthey were not significantly different (p=0.17). This indicates that thevaccination with αGal⁽⁻⁾ B16/NeoR has no impact in the treatment ofsubcutaneous pre established melanoma tumors. On the contrary micereceiving αGal⁽⁺⁾ B16 vaccines, developed smaller tumors that grewslower (n=33). There was a statistically significant difference in thetumor growth of this group compared to the control groups (p<0.03).

These two experiments indicated that the vaccination with αGal⁽⁻⁾ B16cells is not able to treat pre-established subcutaneous melanoma tumors.On the other hand, pre-established subcutaneous melanoma tumors can besuccessfully treated by vaccination with αGal⁽⁺⁾ B16 vaccines.

Survival Analysis of Mice with Pre-Established Subcutaneous TumorsTreated with αGal⁽⁺⁾ B16 Vaccines

Mice bearing pre-established αGal⁽⁻⁾ B16 subcutaneous tumors vaccinatedwith αGal⁽⁺⁾ B16 irradiated cells showed prolonged survival whencompared with non-vaccinated controls and αGal⁽⁻⁾ mock-vaccinated groups(FIG. 19). While zero out of 9 and only 1 out of 20 non-vaccinated andmock-vaccinated animals survived the subcutaneous challenge,respectively, 5 out of 19 mice treated with αGal expressing B16 cellssurvived for more than 70 days after the challenge. The median survivaltime for the no-vaccine and mock vaccine group were 27 and 26 daysrespectively. On the contrary, the median survival time of micereceiving αGal⁽⁺⁾ B16 vaccines was significantly increased (39 days).This result demonstrates that αGal⁽⁺⁾ B16 vaccines cells can efficientlytreat pre-established subcutaneous melanoma tumors demonstrated by theincreased number of surviving animals (26% vs. 5%) and by the prolongedmedian survival time (39 vs. 26 days).

In a second experiment mice received three doses of cell vaccines at 4,11 and 18 days after the initial subcutaneous injection withnon-irradiated αGal⁽⁻⁾ B16 (FIG. 20). The vaccine dose each time was3×10⁵ cells. Mice were either not vaccinated (n=12), vaccinated withαGal⁽⁻⁾ B16 (n=23) or vaccinated with αGal⁽⁺⁾ B16 (n=26).

FIG. 20 shows the Kaplan-Meier survival analysis after 70 days ofobservation. The logrank test comparison of the survival curvesindicated a significant difference in the number of surviving micebearing subcutaneous melanoma tumors treated with αGal⁽⁺⁾ B16 vaccines,compared to control non-vaccinated and αGal⁽⁻⁾ B16 vaccinated mice(p<0.005). None out of 12 non-vaccinated mice survived the subcutaneousinjection with B16. Similarly, none out of 23 mice vaccinated withαGal⁽⁻⁾ B16 vaccines survived the subcutaneous injection with B16. Onthe contrary 11 out of 26 mice (42%) receiving αGal⁽⁺⁾ B16 vaccinessurvived for 70 days after the lethal subcutaneous injection of αGal⁽⁻⁾B16 melanoma.

The median survival of control and mock vaccinated mice were notsignificantly different (38 and 42 days, respectively, logrank testp=0.43, ns). On the contrary the median survival of mice treated withαGal⁽⁺⁾ B16 was greater than 60 days. This represent a significantincrease in the median survival of the αGal vaccinated group (p<0.005).

This result further demonstrated that the treatment of pre-establishedsubcutaneous melanoma tumors with αGal⁽⁺⁾ B16 vaccines is significantlyeffective in comparison to αGal⁽⁻⁾ vaccination or no treatment.

Example 8 Treatment of Lung Melanoma Metastases by αGal⁽⁺⁾ B16 CellVaccines

We further evaluated the efficacy in the treatment of lung melanomametastasis as a model of disseminated disease. This experiment is veryrelevant from the clinical point of view since patients bearing tumorsmost likely will die from disseminated disease, which is surgicallynon-removable. Thus, RRBC-immunized mice were intravenously (i.v)challenged with 10⁵ non-irradiated αGal⁽⁻⁾ B16 and randomized. Mice weretreated subcutaneous with irradiated B16/NeoR vaccine (αGal⁽⁻⁾, n=6) orwith αGal⁽⁺⁾ B16 vaccine (n=7). Vaccines (2×10⁵ irradiated cells) wereadministered subcutaneous at 4, 11 and 21 days after i.v. injection ofB16 (FIG. 21). Mice were sacrificed 30 days after challenge and thenumber of melanoma metastasis were enumerated (FIG. 22). The number oflung metastasis were “too numerous to count” (arbitrary value >250tumors) in 3 mice and 30 tumors were counted in the other mice, whileonly two mice were tumor free. Moreover, one of the animals in thisgroup showed three additional metastatic nodules in the peritonealcavity demonstrating dissemination of the disease in other placesbesides lungs.

On the contrary, mice treated with αGal⁽⁺⁾ B16 vaccine were all tumorfree demonstrating that pre-established tumor were treated verysuccessfully by the αGal⁽⁺⁾ expressing vaccine therapy.

In a second independent experiment (FIG. 23) a five-fold increase doseescalation of non-irradiated αGal⁽⁻⁾ B16 was used. Mice were injectedi.v. with 5×10⁵ live B16 to pre-establish the lung melanoma metastases.Four days later vaccination with control and αGal⁽⁺⁾ B16 cells wasperformed as before. Mice received either no vaccine treatment (n=10),three doses of αGal⁽⁻⁾ B16 (n=11) or αGal⁽⁺⁾ B16 vaccines (n=11).

Thirty days after the i.v. challenge with B16, mice were euthanized andlung melanoma metastases enumerated (FIG. 23). Also the tumor growth wasevaluated by counting the lung tumor burden and by weighting lung tissue(FIG. 24).

Numerous lung melanoma metastases were found in the lungs ofnon-vaccinated mice as well as in the control vaccinated group. Toperform statistic comparisons, the weight of lung tissue was used. Thedifference between the lung burden in control and non-vaccinated groupswas not statistically different (Unpaired t Test p=0.66, ns). On thecontrary, significantly reduced lung burden was observed in micevaccinated with αGal expressing B16 vaccines (One way ANOVA p<0.006).

Similar to the observations of the first experiment, some mice fromcontrol and no-vaccine group had “too numerous to count” melanomametastases. In addition, two animals had scattered melanoma tumorsextra-pulmonary, indicating disseminated disease in addition topulmonary tissue. None of the αGal⁽⁺⁾ B16 vaccinated mice had “toonumerous to count” melanoma tumors in the lungs and none hadextra-pulmonary tumors. This indicates that vaccination with αGal⁽⁺⁾ B16vaccines can effectively treat disseminated metastatic melanoma. Inaddition, the vaccination with αGal⁽⁺⁾ B16 vaccines can prevent furtherspreading of the systemic disease.

Example 9 T Cell Studies to Demonstrate Induction of αGal⁽⁻⁾B16-Specific T Cell Precursors After Vaccination with αGal⁽⁺⁾ B16 Cells

One of the fundamental questions of this technology is whether thevaccination with αGal⁽⁺⁾ cells will induce T cell mediated immunitycapable to react against the αGal⁽⁻⁾ tumor. In the experiments shownabove we demonstrated that αGal⁽⁺⁾ B16 vaccines induce strong immunityable to mediate the clearance of pre-established αGal⁽⁻⁾ tumors. Thisimmunity in not induced when mice are vaccinated with αGal⁽⁻⁾ B16vaccines. To further demonstrate that T cell precursors specific for theαGal⁽⁻⁾ B16 tumors were induced after vaccination with αGal⁽⁺⁾ B16vaccines, in vitro T cell studies were conducted. The goal of theseexperiments was to demonstrate specific recognition of the αGal⁽⁻⁾ B16cells by T cells. T cells were harvested from mice vaccinated withcontrol vaccine αGal⁽⁻⁾ B16 group or from mice vaccinated with αGal⁽⁺⁾B16 irradiated cells (FIG. 25). Two types of studies were performed thatdemonstrated by different means the same conclusion, that is, that micevaccinated with irradiated αGal⁽⁺⁾ B16 cell have increased numbers of Tcell precursors able to recognize specifically αGal⁽⁻⁾ B16 tumor cells.

In the first set of experiments, the intracellular cytokine TNF-α wasdetected by FACS. Cells harvested from mice vaccinated with irradiatedαGal⁽⁻⁾ B16 cells or with αGal⁽⁺⁾ B16 cells, two weeks after the lastsubcutaneous vaccination. Splenocytes were cultured without stimulationas negative control. For stimulation, they were co-cultured with CA320M(αGal⁽⁻⁾ syngeneic H-2 b/b haplotype) as negative control or withαGal⁽⁻⁾ B16 cells. After 6 hours of stimulation cells were harvested andstained for intracellular TNFα. Increased percentage of TNFα⁽⁺⁾lymphocytes was found in the spleens of mice vaccinated with αGal⁽⁺⁾ B16cells that specifically recognized αGal⁽⁻⁾ B16 cells (FIG. 26). These Tcells did not produce TNFα when cultured with CA320M which indicatesthat they specifically recognize B16 and not a non-related syngeneiccell line. In addition to the increased number of melanoma specific Tcell precursors, the quantitative amount of TNFα produced by these cellswas significantly increased measured by the Mean fluorescence intensitydetected by FACS (FIG. 26). Four-fold increased MFI in the TNFα⁽⁺⁾ cellswas detected. When splenocytes from mock-vaccinated mice were culturedwith αGal⁽⁻⁾ B16, only 4% of TNFα⁽⁺⁾ cells were detected with a MFI of17. On the contrary, when T cells from mice receiving αGal⁽⁺⁾ B16 cellvaccines were cultured with αGal⁽⁻⁾ B16, 7% of TNFα⁽⁺⁾ cells weredetected with a MFI of 69. This represents a two fold increase in thepercentage of T cell precursors present in the spleen. This experimentwas repeated a total of three times and similar results were obtained.

This result demonstrated that the vaccination with αGal⁽⁺⁾ B16 cells andnot vaccination with αGal⁽⁻⁾ B16 induces a strong specific T cellimmunity detected in vitro by T cell specific recognition of themelanoma target.

In a different set of experiments, cell-surface activation markers wereused to measure specific T cell recognition of the αGal⁽⁻⁾ B16 melanomacell line. It is well described that upon engagement of the T cellreceptor (TCR), T cells up-regulate several cell surface molecules thatindicate an activated state of the lymphocyte. One of those molecules isthe IL-2 receptor alpha chain or CD25. Upon TCR engagement, CD25 isup-regulated and can be detected by FACS at 1 day after activation.Similarly, CD69 (or very early activation antigen (VEA)) is up-regulatedupon T cell activation. CD69 functions as a signal-transmitting receptorin different cells, it is involved in early events of lymphocyteactivation and contributes to T cell activation by inducing synthesis ofdifferent cytokines, and their receptors. Both activation markers (CD25and CD69) are expressed at very low level in resting T cells. Todemonstrate that vaccination with αGal⁽⁺⁾ B16 cells induced T cellprecursors able to recognize specifically αGal⁽⁻⁾ B16, the up-regulationof activation markers was used as parameters to measure recognition andactivation. Cells were harvested from mice vaccinated with αGal⁽⁻⁾ B16or vaccinated with αGal⁽⁺⁾ B16. They were cultured without stimulationor stimulated with a negative control cell line (CA320M) or with αGal⁽⁻⁾B16. After 24 hours of culture, cell were harvested and stained todetect CD25 or CD69. Acquisition was performed gating in cells excludingthe vital staining 7-AAD (live cells). As expected, resting T cells (nostimulation) and cells stimulated with the syngeneic non-melanoma cellline CA320M expressed very low levels of activation markers (FIGS. 27 aand 27 b). When splenocytes from mice receiving αGal⁽⁻⁾ B16 werecultured with B16, some up-regulation of activation markers wasobserved. This corroborate previous reports from the literature whichindicated that low-degree of immune reactivity can be observed when micereceive native αGal⁽⁻⁾ B16 vaccines. However this reactivity is notsufficient to prevent and or treat pre-established melanoma tumors. Onthe other hand, increased activation of lymphocytes from mice vaccinatedwith αGal⁽⁺⁾ B16 was detected when T cells were cultured with αGal⁽⁻⁾B16, as increased number of CD25⁽⁺⁾ and CD69⁽⁺⁾ cells were measured.

This result once again demonstrated that vaccination with αGal⁽⁺⁾ B16cells induced T cell precursors able to recognize specifically αGal⁽⁻⁾B16 melanoma cells.

Example 10 Treatment of Pre-Established Metastatic Melanoma Tumors byAdoptive T Cell Transfer from Mice Vaccinated with αGal⁽⁺⁾ or αGal⁽⁻⁾B16 Cell Vaccines

The in vitro experiments shown above demonstrated that more quantity andquality of melanoma specific T cells are induced in mice vaccinated withαGal⁽⁺⁾ B16 cells when compared to mice receiving αGal⁽⁻⁾ B16vaccination. These melanoma specific T cells were increased in numbers(more T cells found in spleens) and they produced more TNFα. Also, moresplenocytes were activated when co-cultured with B16 (up-regulation ofCD25 and CD69). In experiments shown before, mice bearing bothsubcutaneous and lung pulmonary metastasis receiving αGal⁽⁺⁾ B16 showedprolonged survival and increased clearance of the lung tumors. Havingthese two groups of data, we could infer that in fact T cells induced byαGal⁽⁺⁾ B16 vaccination are responsible for the treatment ofpre-established melanoma tumors. However, it is not obvious that this isthe case since it has been shown that large amount of melanoma-specificT cells are insufficient to treat pre-established subcutaneous melanomatumors, since they are in a tolerant state [Overwijk et al. “Tumorregression and autoimmunity after reversal of functionally tolerantstate of self-reactive CD8+ T cells” J. Exp. Med. (2003) 198: 569-580].We hypothesized that vaccination with αGal⁽⁺⁾ B16 cells induced a strongcell mediated immunity that can be rapidly activated upon recall and itis responsible for tumor clearance in mice bearing pre-establisheddisease. To demonstrate this hypothesis adoptive cell transferexperiments were conducted (FIG. 28). Donor mice were vaccinatedreceiving three doses of irradiated αGal⁽⁺⁾ B16 or αGal⁽⁻⁾ B16 vaccinesas described before. Recipient mice were i.v injected with live αGal⁽⁻⁾B16 to establish the lung melanoma metastasis and randomized. Four daysafter i.v injection of non-irradiated B16, mice received, or not T cellsfrom donors vaccinated with αGal⁽⁺⁾ or αGal⁽⁻⁾ B16 cells. Four weekslater, the lung melanoma metastasis burden was measured by enumeratinglung tumors, and by weighting lungs obtained in block. The experimentwas performed twice and results from both are depicted in FIGS. 29A and29B. Experiment #1 shows the average lung weight of mice receiving i.vB16 with no T cell therapy (n=16), mice receiving T cells from αGal⁽⁻⁾B16 vaccinated mice (n=15) and mice receiving T cells from micevaccinated with αGal⁽⁺⁾ B16 (n=17). The bars represent the average inlung weight and error bars, the SEM. Large tumors and significantincreased lung burden was demonstrated in control mice and in micereceiving T cells from mock-vaccinated mice. The difference in lungmelanoma metastasis between mice receiving no T cells (control) and micereceiving T cells from mock-vaccinated mice was found not statisticallydifferent (p>0.05). On the contrary significant reduction of the lungmelanoma burden was observed in mice receiving T cells from αGal⁽⁺⁾ B16vaccinated mice (One-way ANOVA p<0.05). Similar results were observed ina second independent experiment. Mice receiving no T cells have anaverage of 64 lung melanoma tumors (n=7). Mice receiving cells fromαGal⁽⁺⁾ B16 vaccinated mice had as many lung melanoma metastasis(mean=90, n=10, p=>0.05). In contrast, an average of only 31 lungmelanoma tumors were observed in mice receiving splenocytes from micevaccinated with αGal⁽⁺⁾ B16 cells. This represents a significantreduction in the lung melanoma burden of mice receiving T cells fromαGal⁽⁺⁾ B16 vaccinate mice (Unpaired t Test p<0.05).

This remarkable success in the reduction of pre-established lungmelanoma metastasis with the sole treatment of cells from αGal⁽⁺⁾vaccinated mice, demonstrates for the first time that strong cellmediated immunity is induced by αGal⁽⁺⁾ cell vaccines and not withαGal⁽⁻⁾ vaccines. This strong cell dependent tumor immunity is, withlittle doubt, responsible for the treatment of disseminatedpre-established disease.

Example 11 Survival of αGal⁽⁻⁾ Knockout Mice Vaccinated with αGal⁽⁻⁾ orαGal⁽⁺⁾ Irradiated CA320M Sarcoma Cells After Subcutaneous Injection ofa Lethal Dose of Non-Irradiated αGal⁽⁻⁾ CA320M Cells

The prophylactic vaccination experiments described above with B16melanoma cells were repeated with a different tumor cell line derivedfrom the αGal⁽⁻⁾ knockout mice and therefore are completely syngeneicwith the host. This cell line is CA320M and was obtained byintraperitoneal injection of 2 mg 9,10-dimethyl-1,2-benz-anthracene(DMBA) and 1 mg 3-methylcholanthrene (3-MC) dissolved in 250 μl of oliveoil at two week intervals into αGT knockout mice. One mouse presented atumor eight months after the first injection. The tumor was localized tothe intraperitoneal cavity with a large mass approximately 2 cm wide by3 cm tall and 1.5 cm deep. Metastatic nodules located on the mesentarieswere noted. Both the primary mass and the intestinal tract werecollected for culture and histopathological examination. Metastaticnodules collected from the mesenteries associated with the primary siteof tumor were successfully cultured and designated CA320M.Histopathological examination of both frozen and paraffin sections ofthe primary and transplanted tumors, respectively, demonstratedmorphologies consistent with a poorly differentiated sarcoma.Hematoxylin and eosin staining along with immunohistochemical analysissuggests that CA320M can be classified in the family of gastrointestinalstromal tumors as a sarcoma of the small intestine. CA320M cells inducedin a αGT KO mouse failed to bind IB₄ isolectin that specifically detectsαGal epitopes, verifying that this murine tumor is, as expected, devoidof functional αGT enzyme and, therefore, αGal epitopes. CA320M cellswere susceptible to infection by an HSV-1 based vector and expressedαGal epitopes after transduction with HE7αGall.

αGT KO mice were primed to develop immunity against the αGal epitope bysubcutaneous injection of 2×10⁷ rabbit whole blood cells twice at 14 dayintervals. CA320M cells were transduced with 10 MOI of either HDKgal orHDKgalΔsal1 for eight hours to obtain CA320M αGal⁽⁺⁾ or αGal⁽⁻⁾ cells,respectively. Briefly, HDKgal was developed by inserting the αGT genewith a Kozak sequence into the pHD1 amplicon HSV-1 vector. pHDKgaltherefore carries a single eukaryotic gene, αGT, under the control ofthe CMV promoter. To construct the mutant αGT gene, a unique SalIrestriction site, located in the corresponding catalytic domain of theαGT enzyme, was cut and filled in using Klenow and dNTP's. The resultingframeshift mutation yields premature termination of the polypeptiderendering the enzyme nonfunctional. The mutant αGT gene was also clonedinto the pHD1 amplicon and both amplicons were packaged into infectiousHSV virions using a helper-free herpes viral system. Transduced CA320Mcells were irradiated (25 Gy) and 1×10³ cells injected into each of 15animals. Twenty-one days later animals were challenged with 1×10⁷ liveCA320M cells and followed for tumor growth and survival analysis.Ninety-three percent of αGal⁽⁺⁾ CA320M vaccinated animals survivedlethal tumor challenge for up to 60 days (>400 mm³ tumor) compared toanimals vaccinated with the mutant αGal⁽⁻⁾ CA320M (33%) and null (38%)controls (FIG. 30). This results confirms that vaccination with αGal⁽⁺⁾syngeneic cells is capable to induce a protective immune responseagainst the corresponding αGal⁽⁻⁾ cells.

Taking together, all these results demonstrate for the first time thatthe presence of α-Galactosyl epitopes in whole cell vaccines represent astrong immune adjuvant able to induce T cell mediated immunity to treatpre-established tumors lacking the expression of αGal epitopes. Theseresults also indicate that clinical use of this vaccine may have asignificant impact in human medicine. In addition, the preventivevaccination for cancer patients currently is not possible consequentlytherapeutic approaches like the technology described here have arealistic value for the rapid application and possible benefit of cancerpatients.

Example 12 Development of Human-Whole Cell Cancer Vaccine (HyperAcute™)

Hyperacute™ whole cell cancer vaccines consists of allogeneic cancercell lines genetically engineered to express murineα(1,3)galactosyltransferase gene. Several independent cancer cell linesfrom several tumor tissue types have been engineered to express αGalepitopes on their surface. A therapeutic whole cell cancer vaccineconsists of injection of irradiated individual cell lines or a mixtureof several engineered cancer cell types belonging to the same tumortissue type. Table 1 indicates the cancer cell lines that were used tooriginate the HyperAcute™ whole cell cancer vaccines.

TABLE 1 HyperAcuteα ™ whole cell cancer vaccines HyperAcute ™ TumorVaccine Cell Line ATCC # Tissue Type Description HAL1 A549 CCL-185 lungcarcinoma HAL2 NCI-H460 HTB-177 lung large cell carcinoma HAL3 NCI-H520HTB-182 lung squamous cell carcinoma HAB1 MCF-7 HTB-22 breast pleuraleffusion adenocarcinoma HAB2 BT-20 HTB-19 breast carcinoma HAPA1 HPAF-IICRL-1997 pancreas ascitis adenocarcinoma HAPA2 PANC-1 CRL-1469 pancreasprimary ductal epithelial carcinoma HAPA3 ASPC-1 CRL-1682 pancreasmetastatic adenocarcinoma HAPA4 BxPC3 CRL-1687 pancreas primaryadenocarcinoma HAO1 IGROV NA ovary carcinoma HAO2 ES-2 CRL-1978 ovaryclear cell carcinoma HAO3 NIH: OVCAR3 HBT-161 ovary ovarianadenocarcinoma HAO4 PA-1 CRL-1572 ovary ovarian teratocarcinoma HAM1A375 CRL-1619 melanoma malignant melanoma HAM2 COLO829 CRL-1974 melanomamalignant melanoma HAM3 G-361 CRL-1424 melanoma malignant melanoma HAC1HCT-116 CCL-247 colon colorectal carcinoma HAC2 COLO205 CCL-222 colonmetastatic adenocarcinoma HAC3 LoVo CCL-229 colon metastaticadenocarcinoma HAC4 WiDr CCL-218 colon colorectal adenocarcinoma HAC5DLD-1 CCL-221 colon colorectal adenocarcinoma HAC6 HCT-15 CCL-225 coloncolorectal adenocarcinoma HAC7 SW620 CCL-227 colon metastaticadenocarcinoma HAC8 SW480 CCL-228 colon metastatic adenocarcinoma HAC9SW1116 CCL-233 colon colorectal adenocarcinoma grade II HAC10 HT-29HTB-38 colon colorectal adenocarcinoma grade II HAC11 FHC CRL-1831 colonnormal mucosal colon HAC12 CCD841CoN CRL-1790 colon normal colon HAPR1PC-3 CRL-1435 prostate metastatic adenocarcinoma HAPR2 LNCaPFGC CRL-1740prostate metastatic adenocarcinoma HAPR3 MDAPca2b CRL-2422 prostatemetastatic adenocarcinoma HAPR4 DU145 HTB-81 prostate metastaticcarcinoma

HyperAcute™ whole cell cancer vaccines were established by transductionof the cell lines indicated in Table 1 with retroviral supernatant from293Z.CKG in the presence of protamine sulfate 10 μg/mL. The populationof transduced cells was stained for αGal cell surface epitopes using0-2605 anti-αGal antibody (NewLink). Five percent of the cell populationwith the highest intensity of αGal epitopes expression on their cellsurface was sorted into a separate subpopulation using FACS sorter. Thesorted subpopulation of transduced cells with highest expression of αGalepitopes was the one designated as indicated in Table 1. These cellswere expanded (split ratio 1:5) to generate a master cell bank (MCB).Growth pattern, morphologic appearance and mean intensity of αGalepitopes expression have been monitored and remained stable during thewhole period of propagation in culture. MCB were developed by expandingthe cells in flasks at 37° C.±1° C. in 5%±1% CO2. The culture medium wasRPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 2 mmL-glutamine. At each passage the cells were trypsinized, counted andtheir viability was assessed by trypan blue exclusion. Cells werepropagated to provide one billion cells, harvested, pooled, distributedinto 100 cryovials, and frozen using programmed rate freezing chamber.Cells are stored in the vapor phase of liquid nitrogen storage tank. Aworking cell bank (WCB) for each cell line was developed from a MCB.Conditions for WCB expansion, harvesting, freezing and storage were asit is described for MCB. A production lot for each HyperAcute™ cancercell line was originated from each WCB. When the cells from theproduction lot reach sufficient density, the culture fluids(supernatant) are harvested, filtered, and pooled into a sterilecontainer. The pool is thoroughly mixed and then aseptically filled intolabeled, sterile plastic bottles. (Labels contain the product name, lotnumber and date of filling). The fill bottles are frozen and stored ator below −60° C. Aliquots are submitted for safety testing. Cells for aProduction Lot are harvested by trypsinization, pooled, and resuspendedin complete culture medium. Pooled cells are irradiated at 150-200 Grey.The cells were irradiated using a Varian Clinic 2100C medical linearaccelerator operating in the 6MV photon mode. The machine's radiationoutput is calibrated in water using the AAPM TG-51 calibration protocol.Output consistency is monitored daily; and output calibration is checkedmonthly with a NIST calibration traceable ion chamber/electrometerdosimeter. Irradiated cells are centrifuged and resuspended in finalformulation for injection consisting of 5% glycerol and human serumalbumin. Then 0.4 ml of cells at concentrations of 1×10⁶/0.2 ml,3.5×10⁶/0.2 ml, 1×10⁷/0.2 ml and 3.5×10⁷ cells/0.2 ml corresponding todose levels I, II, III and IV are distributed into sterile cryovials.The HyperAcute™ cells MCB, WCB, and PL quality control testing involvesboth cells and supernatant. Acceptance criteria for HyperAcute™ cancervaccine MCB, WCB and PL cells consist in assays for tumorigenicity innude mice.

Example 13 Dose Levels and Dosing Schedule for Human Patients

The following data support proposed dose levels and vaccinationschedule. Pre-clinical toxicology studies in mice have shown that α-galexpressing allogeneic and syngeneic tumors are well tolerated up to1×10⁶ cells per mouse which is equivalent to 3.5×10⁹ cells per 70 kghuman. Phase I study with murine vector producer cells (see Section 2.1)naturally expressing α-gal epitopes on cell surface has shown thatmurine VPC were well tolerated at the close 7×10⁹ cells per patient. InPhase II murine VPC trial a patient has undergone three cycles oftreatment with 7×10⁹ cells injected in each cycle. The patient toleratedthe treatment well. Murine VPC injections were repeated with an intervalof 6 weeks. The proposed dose levels are within well-tolerated doses ofα-gal vaccine in mice (maximum 4×10⁸ cells per patient). Four weekintervals between vaccine injections will allow sufficient time forevaluation of toxicity of the treatment and for maturation of the immuneresponse. Data from Phase I murine VPC study suggest that anti-αGalimmune response reaches its maximum between 14 and 21 days after αGalexpressing cells injection.

Example 14 Toxicology Studies Using Syngeneic Tumor Vaccines Transducedwith Retroviral Vector Carrying Murine αGT Gene

In a first study, mice were injected subcutaneously with αGal⁽⁺⁾ B16melanoma cells at a dose of 1 10⁵ cells per mice. Animals that rejectedαGal⁽⁺⁾ living melanoma cells were re-challenged with αGal⁽⁻⁾ B16 cellsto prove that the rejection of αGa⁽⁺⁾ melanoma cells induced tumorimmunity. All mice survived the second challenge with B16. Thesemelanoma-protected animals (n=8 in the first experiment and n=9 in thesecond experiment) were followed for long term toxicity studies for aperiod of six months. Histological, hematological, clinical observationsfor toxicity studies have been performed with some of the protectedmice. Histopathological examinations included major perfused organs:kidney, spleen and liver, as well as potential target tissue, skin andmammary gland. Histological studies evaluating safety indicated noremarkable lesions in all sample tissues examined (skin, mammary gland,kidney, spleen, liver). Some animals (that included control mice) showedrenal perivasculitis with minimal infiltrates of inflammatory cells. Thefrequency of this observation in the test group was similar to thefrequency in the control group of animals. Hematology results showed allvalues studied were within the range of normal values. Normal valueswere considered results from naïve un-manipulated α-gal KO mice Clinicalobservations including behavior, and the development of autoimmunedepigmentation (vitiligo) or fur changes, as a secondary possibleadverse event were not observed during the period of study in melanomaprotected mice (n=17).

In a second study, mice were injected subcutaneously with allogeneicαGal⁽⁺⁾ EMT-6 breast cells at a dose of 1×10⁶ cells per mouse. Two, fourand six weeks after vaccination, blood and tissue samples were obtainedto perform toxicology studies. Histological and hematological toxicitystudies were performed. Histopathology examinations included majorperfused organs: kidney, spleen and liver, as well as potential targettissue, skin and mammary gland. Histological studies evaluating safetyindicated no remarkable lesions in all sample tissues examined (skin,mammary gland, kidney, spleen, liver). Some animals (that includedcontrol mice) showed renal perivasculitis with minimal infiltrates ofinflammatory cells. The frequency of this observation in the test groupwas similar to the frequency in the control group of animals. Hematologyresults showed all values studied were within the range of normalvalues. Normal values were considered results from naïve un-manipulatedα-gal KO mice. Transient eosinophilia was observed at two weeks afterallogeneic vaccination. The maximal dose used in the toxicology studieswas 1×10⁶ cells/mouse. Scaling of this dose to humans based on thedose/kg, assuming that a mouse is 20 grams, shows that the dose/mouse isequivalent to 3.5×10⁹ cells in a 70 kg human. Also considering that theaverage life span of a mouse is about 2 years compared to an average of70 years for humans, the long-term toxicity study (6 months) would beequivalent to one quart of a mice life span, which are about 17 humansyears. These studies support the current dose-schedule 4×10⁸ per patientover a 16-week treatment period.

Example 15 Preparation and Schedule of Administration of HyperAcute™Cancer Vaccine to Patients

This procedure describes how to prepare and administer to human patientsthe whole cell cancer vaccine (the pharmaceutical composition of theinvention). Cells should be injected into patients immediately afterthey have been prepared. No specific safety precautions are necessarybecause administered cells have been lethally irradiated beforefreezing. First, the cryovials of each vaccine cell line are retrievedfrom the liquid nitrogen container. Then, all the vials are thawedsimultaneously by immersing in the water bath at 37° C. to above thefrozen content level. As soon as vials are thawed, rinse their surfacewith 70% alcohol. Equal amounts of content of vial(s) with each cellline component of the vaccine are combined into one syringe forinjection and immediately injected into the patient. The vaccine cellswill be injected intradermally (i.d.) using a tuberculin syringe with a25-gauge needle. Injections should be given in the arms and legs on arotating basis. HyperAcute™ (HAL, HAB, etc.) vaccine cells will beadministered on days 1, 29, 57, and 85. Patients will be monitored fortwo hours after each injection in the outpatient clinic by the nursingstaff. Patient monitoring is to include: Temperature (T), pulse (P),blood pressure (BP) and respiratory rate (R), within 30 minutes beforeadministration of the vaccine, and then by checking every 15 minutes×4,then every 30 minutes×2 after the vaccination. Temperature will bechecked prior to discharge from the clinic. In addition, patients willbe monitored for signs of acute reactions including local ordisseminated skin rash and other adverse reactions. Patientsexperiencing Grade II or greater acute adverse events may be monitoredfor an additional 1-2 hours in the clinic until the event has resolvedto less than Grade II. If an AE of Grade II or greater persists for morethan 4 hours despite observation and/or treatment, a decision on whetherto continue observation, institute or modify treatment, or admit thepatient to the hospital

Example 16 Determination of Maximum Tolerated Dose

Treatment will proceed in dose cohorts of 3 eligible patients. Themaximum tolerated dose (MTD) is defined as the dose cohort below that atwhich dose-limiting toxicity (DLT) is seen. If >33% of patients (i.e.,⅓, or 2/4-6) in a dose cohort manifest DLT, then the MTD will have beendetermined and further dose escalations will not be permitted. If DLT isnoted in one of three patients (⅓) in a dose cohort, then that cohortwill be expanded to accrue up to a total of six (6) patients. If anotherDLT is observed, the cohort will be closed, MTD will be defined and nofurther dose escalation permitted. If no other DLT is observed in thecohort, accrual to the next higher dose cohort will be initiated.Further patients will be treated on the Phase II portion of the study atthe MTD.

There will be a minimum delay of 4 weeks between the entry of the lastpatient on a dose cohort and the entry of the first patient on the nexthigher dose cohort.

TABLE 2 Treatment Plan Cohort Pts. (N = 3) Vaccine HAL Cells A 3 3 × 10⁶B 3 1 × 10⁷ C 3 3 × 10⁷ D 3  1 × 10⁸* *Injection site may be split dueto number/volume of cells used.

Response and progression is evaluated in this study using the newinternational criteria proposed by the Response Evaluation Criteria inSolid Tumors (RECIST) Committee. Changes in only the largest diameter(unidimensional measurement) of the tumor lesions are used in the RECISTcriteria. Lesions are either measurable or non-measurable using thecriteria provided below. The term “evaluable” in reference tomeasurability will not be used because it does not provide additionalmeaning or accuracy. Measurable lesions are defined as those that can beaccurately measured in at least one dimension (longest diameter to berecorded) as ≧20 mm with conventional techniques (CT, MRI, x-ray) or as≧10 mm with spiral CT scan. All tumor measurements must be recorded inmillimeters (or decimal fractions of centimeters). All other lesions (orsites of disease), including small lesions (longest diameter <20 mm withconventional techniques or <10 mm using spiral CT scan), are considerednon-measurable disease. Bone lesions, leptomeningeal disease, ascites,pleural or pericardial effusions, lymphangitis cutis or pulmonis,inflammatory breast disease, abdominal masses (not followed by CT orMRI), and cystic lesions are all considered non-measurable. Allmeasurable lesions up to a maximum of five lesions per organ and 10lesions in total, representative of all involved organs, should beidentified as target lesions and recorded and measured at baseline.Target lesions should be selected on the basis of their size (lesionswith the longest diameter) and their suitability for accurate repeatedmeasurements (either by imaging techniques or clinically). A sum of thelongest diameter (LD) for all target lesions is calculated and reportedas the baseline sum LD. The baseline sum LD will be used as reference bywhich to characterize the objective tumor response. All other lesions(or sites of disease) should be identified as non-target lesions andshould also be recorded at baseline. Non-target lesions includemeasurable lesions that exceed the maximum numbers per organ or total ofall involved organs as well as non-measurable lesions. Measurements ofthese lesions are not required but the presence or absence of eachshould be noted throughout follow-up. All measurements should be takenand recorded in metric notation using a ruler or preferably calipers.All baseline evaluations should be performed as closely as possible tothe beginning of treatment and never more than 2 weeks before thebeginning of the treatment. Tumor lesions that are situated in apreviously irradiated area are not considered measurable. Clinicallesions are only be considered measurable when they are superficial(e.g., skin nodules and palpable lymph nodes). In the case of skinlesions, documentation by digital color photography, including a rulerto estimate the size of the lesion, is recommended. Lesions on chestx-ray are acceptable as measurable lesions when they are clearly definedand surrounded by aerated lung. However, CT is preferable. Conventionalcomputed tomography (CT) and magnetic resonance imaging (MRI) should beperformed with cuts of 10 mm or less in slice thickness contiguously.Spiral CT should be performed using a 5 mm contiguous reconstructionalgorithm. This applies to tumors of the chest, abdomen, and pelvis.Head and neck tumors and those of extremities usually require specificprotocols. Ultrasound (US) is utilized when the primary endpoint of thestudy is objective response evaluation. US should not be used to measuretumor lesions. It is, however, a possible alternative to clinicalmeasurements of superficial palpable lymph nodes, subcutaneous lesions,and thyroid nodules. US might also be useful to confirm the completedisappearance of superficial lesions usually assessed by clinicalexamination. Endoscopy and Laparoscopy is for objective tumor evaluationhas not yet been fully and widely validated. Their uses in this specificcontext require sophisticated equipment and a high level of expertisethat may only be available in some centers. Therefore, the utilizationof such techniques for objective tumor response should be restricted tovalidation purposes in reference centers. However, such techniques canbe useful to confirm complete pathological response when biopsies areobtained. Tumor markers-alone cannot be used to assess breast tumorresponse. Cytology and histology can be used to differentiate betweenpartial responses (PR) and complete responses (CR) in rare cases (e.g.,residual lesions in tumor types such as germ cell tumors, where knownresidual benign tumors can remain). Cytopathological confirmation of theneoplastic origin of any effusion that appears or worsens duringtreatment when measurable tumor has met the criteria for response orstable disease is mandatory to differentiate between response or stabledisease (an effusion may be a side effect of the treatment) andprogressive disease.

Example 17 Phase Study of an Antitumor Vaccination Usingα(1,3)galactosyltransferase Expressing Allogeneic Tumor Cells inPatients with Refractory or Recurrent Non-Small Cell Lung Cancer (NSCLC)

Lung cancer remains the leading cause of cancer death. In a phase I/IItrial, we examined the safety and feasibility of antitumor vaccinationin patients with advanced non-small cell lung cancer (NSCLC) withgenetically altered allogeneic human lung cancer cells engineered toexpress xenotransplantation antigens by retroviral transfer of themurine α(1,3) galactosyltrasferase gene (HyperAcute™ Lung CancerVaccine). Patients with stage IV, recurrent or refractory NSCLC, ECOG PS≦2, prior chemotherapies ≦2, adequate bone marrow and organ function,and albumin ≧3.0 g/dL were eligible. Cohorts of 3 patients each werescheduled to receive 4 doses of intradermal injections of 3, 10, 30, or100 million vaccine cells every 4-weeks. HyperAcute™ Lung Cancer Vaccineconsisted in a mixture of equal number of gamma-irradiated cell linesHAL1, HAL2 and HAL3. Toxicity was assessed using the CTC v3.0 andresponse determined by RECIST criteria. To date, 7 patients, 5 males, 2females, median age 56 yrs (range, 41-72), median number of priorchemotherapies=1 (range, 1-2) were treated. Four patients have received4/4 vaccinations, 2 patients received 3 vaccinations and 1 patientreceived 2 vaccinations. Adverse events (≦CTC grade 2) definitely,probably or possibly attributable to the vaccine include injection sitepain/discomfort, local skin reaction, fatigue, and hypertension. Otheradverse events (≦grade 2) include bradycardia, cough, diarrhea, dyspnea,headache, hyperglycemia, hyponatremia, nausea, pleural effusion, andvomiting. To date no Grade 3 or 4 adverse events attributed to thevaccine have been observed. One patient had cough syncope (grade 3) thatwas not related to the vaccine. Table 1 shows a synopsis of thepatients, adverse events and response to date. One patient treated atthe lower dose showed progression of the disease and voluntarily droppedoff the study before receiving all 4 doses of the vaccine. Two patientswith stage IIIA of non-small cell lung cancer (NSCLC) that received allfour doses of the lower dose of vaccine (3 million cells per dose)showed stable disease after 7 months of vaccination. Similarly, twopatients with stage IIIB and IV NSCLC, in the second cohort of patientsthat received all 4 doses of the vaccine (10 million cells per dose)also showed stabilization of the disease at 4 months after startingvaccination. Only one patient with stage IV disease has been treated inthe third cohort of patients (2 doses of 30 million cells) so far,showing no adverse effects. In total, four patients had stable diseasefor >16 weeks (range, 17-28 weeks). The median survival of Stage IVNSCLC subjected to several chemotherapeutic regimens is 5.5 to 8 monthsand patients generally show progressive disease during this period.Current treatments have limited impact on outcome. Data of the currentPhase I/II clinical trial indicate that vaccination of patients withstage III-IV NSCLC with HyperAcute™ lung cancer vaccines is safe,feasible and more effective than historical chemotherapeutic treatmentcontrols or historical untreated controls, as stabilization of thedisease was achieved after 4 doses of vaccination in 80% of the patientstreated so far.

From the foregoing it can be seen that the invention accomplishes atleast all of its objectives. All references cited herein are herebyincorporated in their entirety.

TABLE I NLGO101-HyperAcute ™-Lung Cancer Vaccine Trial, IND#11261:Summary of Patients, Results and Adverse Events HAL Vaccine Patient AgeSex NSCLC Cells per Date on Study/No. No. (Yrs.) (M/F) Initial StageDose Vaccines Grade/Adverse Event Response 1 41 M IIIB  3 million 20Apr. 2004/ Grade 1 Fatigue Progressive Disease 3/4 vaccinations Grade 1Bradycardia CNS metastases at 5 *Voluntarily came off Grade 1Hyperglycemia months study after 3rd dose. Grade 1 Cough Grade 1 NauseaGrade 2 Vomiting Grade 2 Pleural effusion Grade 1 Injection sitereaction 2 58 M IIIA  3 million 22 Apr. 2004/ Grade 1 HyperkalemiaStable Disease 4/4 vaccinations Grade 1 Cough Grade 1 Dyspnea Grade 1Fatigue Grade 1 CPK Grade 1 Injection site reaction 3 70 M IIIA  3million 27 May 2004 Grade 1 Injection site reaction Stable Disease 4/4vaccinations Grade 2 Hyperglycemia Grade 1 Diarrhea 4 49 F IIIB 10million 15 Jul. 2004/ Grade 1 Diarrhea Stable Disease 4/4 vaccinationsGrade 2 Headache Grade 1 Injection site reaction Grade 1 HypocalcaemiaGrade 1 Hyponatremia Grade 1 Hypercholesterolemia 5 56 F IV 10 million12 Aug. 2004/ Grade 1 Injection site reaction Stable Disease 4/4vaccinations (acute) Grade 2 Pain, skin Grade 1 Pain-muscle 6 49 M IV 10million 09 Sept. 2004 Grade 1 Injection site Not Assessed Yet 3vaccinations to date Reaction Grade 3 Cough (tussive) syncope 7 72 M IV30 million 14 Oct. 2004 None Not Assessed Yet 2 vaccinations to date

1-39. (canceled)
 40. A pharmaceutical composition comprising a tumorcell preparation obtained from a human tumor cell line, each cellcomprising a plurality of cell surface glycoproteins on which an αGalepitope is present, wherein αGal epitopes are present in an amounteffective to treat a pre-existing tumor, suspended in a pharmaceuticallyacceptable carrier suitable for injection in human patients.
 41. Thepharmaceutical composition of claim 40, wherein said αGal epitopes aresynthesized on the surface of said tumor cells by transfecting saidcells with an α(1,3) galactosyltransferase gene.
 42. The pharmaceuticalcomposition of claim 40, wherein said pre-existing tumor is selectedfrom the group consisting of pancreas, prostate, lung and melanoma. 43.The pharmaceutical composition of claim 42 wherein said pre-existingtumor is a lung tumor.
 44. The pharmaceutical composition of claim 43,wherein said tumor cell line is selected from the group consisting oflung tumor cell lines A549, NCI-H460, NCI-H520 and combinations thereof.45. The pharmaceutical composition of claim 42, wherein saidpre-existing tumor is a pancreatic tumor.
 46. The pharmaceuticalcomposition of claim 45, wherein said tumor cell line is selected fromthe group consisting of pancreatic tumor cell lines HPAF-II, PANC-1,ASPC-1, BxPC3 and combinations thereof.
 47. The pharmaceuticalcomposition of claim 42, wherein said pre-existing tumor is a melanomatumor.
 48. The pharmaceutical composition of claim 47, wherein saidtumor cell line is selected from the group consisting of melanoma tumorcell lines A375, COL0829, G-361 and combinations thereof.
 49. Thepharmaceutical composition of claim 42, wherein said pre-existing tumoris a prostate tumor.
 50. The pharmaceutical composition of claim 49,wherein said tumor cell line is selected from the group consisting ofprostate cell lines PC-3, LNCaPFGC, MDAPca2b, DU145 and combinationsthereof.
 51. The pharmaceutical composition of claim 40, wherein saidcomposition is derived from a master cell bank prepared by sorting asubpopulation of tumor cells having the highest expression of αGalepitopes.